Compounds and methods for trans-membrane delivery of molecules

ABSTRACT

A novel delivery system for drugs, and especially macromolecules such as proteins or oligonucleotides through biological membranes is provided, and specifically delivery of siRNA The delivery system comprises conjugation of the macromolecule drug to a moiety that enables effective passage through the membranes. Respectively, novel compounds and pharmaceutical compositions are provided, utilizing said delivery system. In one aspect of the invention, the compounds may be utilized in medical practice, for example, in delivery of siRNA or antisense oligonucleotides across biological membranes for the treatment of medical disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.14/870,406, filed on Sep. 30, 2015, which is a continuation-in-part ofU.S. application Ser. No. 14/830,799, filed on Aug. 20, 2015, which is acontinuation-in-part of PCT International Application No.PCT/IL2015/000019, International Filing Date Mar. 29, 2015, claiming thebenefit of US Provisional Patent Applications Nos. 61/971,548, filedMar. 28, 2014, 61/978,903, filed Apr. 13, 2014, 62/002,870, filed May25, 2014, 62/008,509 filed Jun. 6, 2014, and 62/091,551, filed Dec. 14,2014, which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to a novel delivery system and methods fordelivery of molecules and macromolecules across biological membranesinto cells, optionally with subsequent intracellular entrapment.

BACKGROUND

Protein pathology is a common denominator in the etiology orpathogenesis of many medical disorders, ranging from malfunction of amutated protein, to pathological gain of function where a specificprotein acquires a novel property, which renders it toxic. Conceptually,inhibition of the synthesis of these of proteins by gene therapy mayhold promise for patients having such protein anomaly.

One of the major advances of recent years is the concept of silencing aspecific gene by RNA interference, using small interfering RNA (siRNA).RNA interference is based on short (≈19-27 base pairs), double-strandedRNA sequences (designated siRNA), capable of acting, in concert withcellular biological systems [among others, the Dicer protein complexwhich cleaves double-stranded RNA to produce siRNA, and the RNA-inducedsilencing complex (RISC)], to inhibit translation and mark fordegradation specific mRNA sequences, thus inhibiting gene expression atthe translational stage. The use of antisense oligonucoleotide (ASO),being a short sequence (usually 13-25 nucleotides) of unmodified orchemically modified DNA molecules, complementary to a specific messengerRNA (mRNA), has also been used to inhibit the expression and block theproduction of a specific target protein.

However, albeit the tremendous potential benefits of such approaches formedical care, delivery of such macromolecules into cells remains asubstantial challenge, due to the relatively large and highly-chargedstructures of oligonucleotides (for example, siRNA has an averagemolecular weight of 13 kD, and it carries about 40 negatively-chargedphosphate groups). Therefore, trans-membrane delivery ofoligonucleotides requires overcoming a very large energetic barrier.

The membrane dipole potential is an electric potential that existswithin any phospholipid membrane, between the water/membrane interfaceand the membrane center (positive inside). It is assumed to be generatedby the highly ordered carbonyl groups of the phospholipid glycerylesteric bonds, and its amplitude is about 220-280 mV. Since the membranedipole potential resides in a highly hydrophobic environment ofdielectric constant of 2-4, it translates into a very strong electricfield of 10⁸-10⁹ V/m. Conceivably, the membrane dipole potential andrelated intra-membrane electric field are highly important for thefunction of membrane proteins, determining the conformation and activityof membrane proteins. However, to the best of our knowledge, to date,the dipole potential has not been recruited for drug development.

Various methods have been developed for delivery of macromolecules suchas oligonucleotides or proteins across biological membranes. Thesemethods include viral vectors, as well as non-viral delivery systems,such as cationic lipids or liposomes. However to date, use of thesemethods has been largely limited to applications in vitro, or to focaladministration in vivo, for example, by direct injection into the eye ordirect administration into the lung. Efficient delivery has also beenachieved to the liver. Among these methods, electroporation is aneffective and widely-used method for delivery of macromolecules invitro. According to this method, an external electric field is appliedto a cell suspension, leading to collision of charged target moleculeswith the cell membrane, subsequent temporary and focal membranedestabilization, and consequent passage of the macromolecules into thecell. However, as described above, electroporation is mainly used invitro. Electroporation in vivo encounters limited success, and wasattempted only to specific organs (e.g., muscle, lung), where externalelectrodes could be inserted into the target organ.

In conclusion, delivery of macromolecules, such as oligonucleotides orproteins through cell membranes, or through other biological barriers,such as the Blood-Brain-Barrier, Blood-Ocular-Barrier orBlood-Fetal-barrier still presents a substantial unmet need, andsystemic delivery of therapeutic macromolecules, still remains a huge,unaddressed challenge.

SUMMARY OF THE INVENTION

Embodiments of the present invention provide a novel delivery system,based on a novel, rationally-designed “Molecular NanoMotors (MNMs)”. TheMNMs according to embodiments of the invention have the structure ofmoiety E, E′ or E″ as set forth in Formula (II) below. The drugs to bedelivered by the MNMs may be small-molecule drugs, or macromoleculessuch as peptides, proteins or oligonucleotides (e.g., single-stranded ordouble-stranded, RNA or DNA). In an embodiment of the invention, themacromolecules to be delivered may include RNA strands for genesilencing, i.e., siRNA (small interfering RNA), or DNA sequencesdesigned to serve as antisense oligonucleotides (ASO).

Conjugates of drugs (e.g., small molecule drugs or macromolecules) withMNMs according to embodiments of the invention may be utilized in basicresearch or clinical medical practice, among others, for treatment ofmedical disorders, where aberrant proteins or protein dysfunction play arole, and where silencing the expression of genes encoding for theseproteins can be beneficial; for example, in the treatment ofdegenerative disorders, cancer, toxic or ischemic insults, infections,or immune-mediated disorders.

Conjugates according to embodiments of the invention have the generalFormula (I):

including pharmaceutically acceptable salts, hydrates, solvates andmetal chelates of the compound represented by the structure as set forthin Formula (I), and solvates and hydrates of the salts, wherein:

-   D is a drug to be delivered across biological membranes. D may be a    small-molecule drug, a peptide, a protein, or a native or modified,    single-stranded or double-stranded DNA or RNA, such as ASO or siRNA;-   y, z and w are each an integer, independently selected from 0, 1, 2,    3, 4, 5, 6, wherein at least one of y, z or w is different from 0.    In one embodiment, y=1, z=o and w=0; in another embodiment y=1, z=1    and w=0.    E, E′ or E″ can be the same or different, each having the structure    as set forth in general Formula (II):    (A)_(a)-B-L₁-Q₁-L₂-Q₂-L₃   Formula (II)    where A is selected from the structures as set forth in Formulae    (III), (IV), (V) and (VI):

-   -   M is selected from —O— or —CH₂—; and g, h and k are each        individually an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8,        9, 10, 11, 12, 13, 14, 15 and 16; * is —H, or the point of        linkage to B; a is an integer, selected from 1, 2, 3 or 4;    -   B is selected from the group consisting of:        -   linear, cyclic or branched C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈,            C₉, C₁₀, C₁₁, C₁₂, C₁₃ or C₁₄, alkyl or hetero-alkyl;        -   linear, cyclic or branched C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉,            C₁₀, C₁₁, C₁₂, C₁₃ or C₁₄ alkylene or heteroalkylene;        -   C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃ or C₁₄ aryl or            heteroaryl;        -   one or more steroid moiety (such as, cholesterol, bile acid,            estradiol, estriol), estrogen, nucleoside, nucleotide; and            any combination thereof;        -   wherein each group is optionally substituted by hydroxyl,            amine, or thiol;

-   Q₁ and Q₂ are each independently an optionally cleavable group,    selected from null, ester, thio-ester, amide [e.g., —C(═O)—NH— or    —NH—C(═O)—], carbamate [e.g., —O—C(═O)—NH— or —NH—C(═O)—O—], urea    [—NH—C(═O)—NH—], disulfide [—(S—S)—], ether [—O—], imidazole,    triazole, a pH-sensitive moiety, a redox-sensitive moiety; a metal    chelator, including its chelated metal ion; and any combinations    thereof;

-   L₁, L₂ and L₃ are each independently selected from null and the    group consisting of:    -   linear, cyclic or branched C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉,        C₁₀, C₁₁, C₁₂, C₁₃ or C₁₄, alkyl or hetero-alkyl;    -   linear, cyclic or branched C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀,        C₁₁, C₁₂, C₁₃ or C₁₄ alkylene or heteroalkylene;    -   C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃ or C₁₄ aryl or        heteroaryl;    -   —(O—CH₂—CH₂)_(u)—, wherein u is an integer of 1, 2, 3, 4, 5;    -   nucleoside, nucleotide; imidazole, azide, acetylene; and any        combinations thereof;    -   wherein each group is optionally substituted by one or more        hydroxyl, amine, or thiol;        wherein each of Q₁, Q₂, L₁, L₂ and L₃ is optionally substituted        by T; wherein T is an initiator group, selected from C₅, C₆,        C₇-1,2-dithiocycloalkyl(1,2-dithiocyclo-pentane,        1,2-dithiocyclohexane, 1,2-dithiocycloheptane); γ-Lactam (5        atoms amide ring), δ-Lactam (6 atoms amide ring) or ε-Lactam (7        atoms amide ring); γ-butyrolactone (5 atoms ester ring),        δ-valerolactone (6 atoms ester ring) or ε-caprolactone (7 atoms        ester ring); and T optionally comprises an amine group.        In another embodiment, there is provided a molecule according to        general Formula (I), which includes E, E′ or E″, having the        structure as set forth in Formulae (XI):

including pharmaceutically acceptable salts, hydrates, solvates andmetal chelates of the compound represented by the structure as set forthin Formulae (XI) and solvates and hydrates of the salts.Some embodiments of the invention relate to a method for delivery of adrug across a biological membrane into cells, either in vitro or invivo, the method comprising contacting the cells with a Conjugate asdescribed herein.

Another embodiment, relates to a method for treating a medical disorderin a patient in need thereof; the method comprises administration to thepatient in need therapeutically efficient amounts of a pharmaceuticalcomposition, comprising a Conjugate as described herein.

In some embodiments of the invention, the medical disorder is cancer.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

The invention will now be described in connection with certain Examplesand embodiments, in a non-limiting manner, with reference to thefollowing illustrative figures, so that it can be more fully understood.In the drawings:

FIG. 1a is a schematic presentation of the principle of asymmetricalpolarity, underlying the putative Mechanism Of Action (MOA) of compoundsaccording to embodiments of the invention;

FIG. 1b schematically depicts structural motifs of the molecules of theinvention, as exemplified by a compound according to Formula (VII),wherein Q₁ is —S—S—; and Q₂ is null;

FIG. 2 schematically illustrates a putative MOA of a conjugate accordingto embodiments of the invention: (i). A “Molecular NanoMotor (MNM)”,energized by the internal membrane electric field, which relates to themembrane dipole potential; (ii). Forced proximity of the macromoleculeto the membrane surface induced by the MNM, forcing lateral movement ofthe phospholipid head-groups; (iii). Subsequent formation of transientmembrane pores, through which there is movement of the macromoleculesinto the cell. This is followed by spontaneous closure of the membranepore and membrane healing.

FIG. 3 schematically illustrates a mechanism for entrapment of siRNAwithin the cytoplasm, utilizing the Dicer enzyme to cleave and removethe MNM; (i). Docking of siRNA, linked to two Apo-Si MNMs on the Dicerprotein; (ii). Removal of one motor by enzyme-mediated RNA cleavage.

FIG. 4 shows an exemplary structure of a Conjugate of the invention,comprising a protein (for example without limitation Cas9) and Emoieties as set forth Formula I;

FIGS. 5a-5f, 6a-c and 7-9 exemplify the biological performance in vitroof conjugates according to embodiments of the invention, comprising MNMsof the invention, having the structure as set forth in either Formula(XI) or Formula (XVI):

FIG. 5a -5 f: 3T3-cells:

FIG. 5a shows fluorescent microscopy of delivery of a Conjugate,comprising a 29-mer, single-stranded DNA (ssDNA) across biologicalmembranes of 3T3 cells, expressing the EGFP Protein (3T3-EGFP cells) invitro;

FIG. 5b shows quantification of the delivery as described in FIG. 5a byflow cytometric analysis (FACS), presented as a dot plot;

FIG. 5c shows quantification by ELISA reader of the delivery asdescribed in FIG. 5a , at 24 hours of incubation;

FIG. 5d shows fluorescent microscopy of delivery of a conjugate,comprising a 58-mer double-strand DNA (dsDNA) across biologicalmembranes of 3T3 cells, expressing the EGFP Protein (3T3-EGFP cells) invitro;

FIG. 5e shows quantification of the delivery as described in FIG. 5d ,by flow cytometric analysis (FACS): (i). Dot plot; (ii). Histogram;

FIG. 5f shows delivery as described in FIG. 5d , detected by confocalmicroscopy, confirming that the delivery of a Conjugate of theinvention, comprising a 58-mer double-stranded DNA is into the cytoplasmof the 3T3-EGFP cells.

FIG. 6a-6c : Murine Melanoma B16 Cells:

FIG. 6a presents fluorescent microscopy of the delivery of a Conjugateof the invention, comprising a 58-mer double-stranded DNA, acrossbiological membranes of B16 melanoma cells in vitro: (i). Control; (ii).A Conjugate comprising MNMs;

FIG. 6b shows quantification of the delivery as described in FIG. 6a ,by flow cytometric analysis (dose/response);

FIG. 6c shows delivery as described in FIG. 6a , detected by confocalmicroscopy, confirming that the delivery of the conjugate, comprising a58-mer double-strand DNA, is into the cytoplasm of the B16 cells.

FIG. 7: Murine C26 Colon Carcinoma Cells:

Flow cytometric analysis of the delivery of a Conjugate of theInvention, comprising a 58-mer double-stranded DNA, across thebiological membranes of C26 cells in vitro.

FIG. 8: HeLa Cells:

Flow cytometric analysis, of the delivery of a conjugate comprising a58-mer double-stranded DNA across the biological membranes of HeLa cellsin vitro; dose/response.

FIG. 9: Gene silencing (EGFP gene), exerted in human HeLA cells by aConjugate of the invention, being a respective siRNA,specifically-designed to silence the EGFP gene, linked to two MNMs, eachhaving the structure as set forth in Formula (XVI) (mean±SEM).

FIG. 10: Mechanism of redox-sensitive cleavage of the Conjugate of theInvention according to Formula (XI), with release of the cargo drug D inthe cytoplasm.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention relate to novel Conjugates,comprising a delivery system for drugs across biological membranes intothe cytoplasm, or through biological barriers, such as, theblood-brain-barrier (BBB), the blood-ocular barrier (BOB), or theblood-fetal-barrier (placental-blood-barrier). Compounds according toembodiments of the invention comprise novel, rationally-designed“Molecular NanoMotors (MNMs)”, rationally-designed to move withinphospholipid membranes, from the membrane/water interface to themembrane core, utilizing the internal membrane electric field, generatedby the membrane dipole potential. When attached to a drug, the deliverysystem acts to re-locate the drug towards the membrane center, thusassisting in its trans-membrane movement. Among others, this deliverysystem is designed for the delivery of therapeutic macromolecules:proteins or oligonucleotides, the latter being single or double-strandedDNA or RNA. Among others, the delivery system is designed for thedelivery of antisense oligonuclotides (ASO), siRNA or therapeuticproteins, such as, for example without limitation, the Cas9 protein orantibodies.

Proposed in a non-limiting manner, one of the principles underlying thestructures of MNMs according to embodiments of the invention is theprinciple of “asymmetrical polarity”. This principle relates tohydrophobic, uncharged molecules, that according to their log P arecapable of partitioning into biological membranes, [for example withoutlimitation, having a log P value>1 (see FIG. 1 A)]. In addition, thesemolecules are polar, and have their partial charges distributed in anuneven manner: the partial negative charge is highly focused andlocalized, while the partial positive charge is dispersed alonghydrocarbon chains within the molecule. Furthermore, upon interactionwith the phospholipid membrane, the partial positive charge is alsomasked, through London type hydrophobic interactions, taking placebetween hydrocarbon chains of the molecule and adjacent hydrocarbonchains of the phospholipid milieu (London dispersion forces).Consequently, as schematically illustrated in FIG. 1A, the molecules ofthe invention are capable of moving in the membrane milieu. Since theinternal membrane electric field has a negative pole at themembrane/water interface, and a positive pole at the membrane center,the molecules of the invention move toward the membrane center, and whenattached to a cargo (e.g., a drug such as siRNA, ASO, a therapeuticprotein or another medicament), the cargo is re-located onto themembrane center. This movement may facilitate the trans-membranemovement of the cargo molecule in several ways. Among others, it mayenforce proximity of a charged macro-molecule to the phospholipidheadgroups (PLHG), perturb the hydration shells around the PLHG, andthus force lateral movement of the PLHG. Formation of transient poreswithin the membrane may then takes place, with passage of the cargo drugthrough these pores into the cell. Subsequent spontaneous closure ofthese transient pores may then take place, thus sealing the membranepore, with membrane healing (FIG. 2).

The Conjugates of the invention may also comprise a cleavable group(e.g., a disulfide group, or an oligonucleotide sequence cleavable bythe Dicer enzyme) (FIG. 1b , or FIG. 3). Cleavage of a Conjugate of theinvention at these sites may act to trap the cargo drug (e.g., highlynegatively-charged siRNA or ASO, or other medicament) in the cytoplasmof the target cell. In addition, the continuous consumption of theConjugate due to its cleavage may also assist in maintaining aconcentration gradient of the Conjugate across the cell membrane. Theterm “cleavable group” in the context of the present invention,therefore relates to a chemical moiety, capable of undergoingspontaneous or enzyme-mediated cleavage in certain physiologicalconditions, such as changes in pH, changes in red-ox state, or otherconditions within cells. Examples for cleavable groups are ester,thio-ester, amide, carbamate, disulfide, ether, a pH-sensitive moiety, aredox-sensitive moiety, or a metal chelator [which thereby includes itschelated metal ion(s)]. Conceptually, a cleavable group may assist inentrapment of a drug within a target cell following its trans-membranepassage, or assist in maintaining a concentration gradient of theConjugate of the invention) across the biological membranes.

For example, in the case of a Conjugate according to an embodiment ofthe invention, that comprises siRNA, ASO or a therapeutic protein aspharmaceutically-active drugs, and a disulfide group as a cleavablegroup, once inside the cytoplasm, the prevailing ambient reductiveenvironment will act to reduce the disulfide bond to free thiol groups,thus cleaving the Conjugate, and leading to disengagement of the MNMsfrom the cargo drug. Devoid of the MNM, the charged cargo macromoleculewill eventually be captured in the cytoplasm, where for example, in thecase of siRNA, it will be ready for interaction with the Diver enzyme,or the RNA-induced silencing complex (RISC), resulting in silencing ofthe expression of a specific gene. According to embodiments of theinvention, the gene may encode for a protein playing a role in theetiology or pathogenesis of a specific disease.

The term “initiator group”, in the context of the present invention,relates to a chemical group, that when it undergoes a spontaneous or anenzyme-mediated chemical reaction, it initiates cleavage of an adjacentchemical bond. In more specific embodiments of the invention, theinitiator group is selected from C₄, C₅,C₆-1,2-dithiocycloalkyl(1,2-dithiocyclo-butane; 1,2-dithiocyclopentane;1,2-dithiocyclohexane; 1,2-dithiocycloheptane); γ-Lactam (5 atoms amidering), δ-Lactam (6 atoms amide ring) or ε-Lactam (7 atoms amide ring);γ-butyrolactone (5 atoms ester ring), δ-valerolactone (6 atoms esterring) or ε-caprolactone (7 atoms ester ring).

The term “activated ester” in the context of the present invention,relates to a derivative of carboxylic acids, harboring a good leavinggroup, and thus being capable of interacting with amines to form amides.An example for such activating agent for carboxylic acid isN-hydroxysuccinimide (NHS).

The term “metal chelator” in the context of the present invention,relates to a chemical moiety that entraps a metal ion throughcoordination, wherein the coordinating atoms are selected from nitrogen,sulfur or oxygen atoms. In a preferred embodiment, the chelated ion(s)is calcium (Ca⁺²), coordinated by nitrogen and oxygen atoms. In anotherpreferred embodiment, the metal chelator is BAPTA[1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid], EGTA(ethylene glycol tetraacetic acid) or analogues thereof, manifestingadvantageous selectivity for Ca⁺² over other ions such as Mg⁺². Suchchelators may enable utilization of the substantial concentrationgradient of Ca⁺² between the extracellular space and the cystosol, forpotential disengagement of the MNM from the cargo drug and capture andaccumulation of the target drug within the cytoplasm.

The term“heteroalkyl, heteroalkylene or heteroaryl” in the context ofthe invention, relates to the respective hydrocarbon structure, where aleast one of the atoms has been replaced by a nitrogen, oxygen, orsulfur atom(s), or any combination thereof.

According to one of the embodiments of the invention, the “cargo” or the“cargo drug” is a siRNA, ASO, a therapeutic protein, or any othermedicament to be delivered across cell membranes and into cells. Saidcells may be either in cell culture of within the body of a livinganimal or a human subject, and said delivery aims at exerting beneficialtherapeutic effects.

The term “precursor” in the context of the invention, relates to achemical moiety, used in the synthesis of conjugates according toembodiments of the invention. The precursor comprises chemical groups,destined to be removed during the synthesis of the Conjugate in variousstages of the synthesis, for example without limitation, during theattachment of a macromolecule, such as an oligonucleotide to MNMs of theinvention.

The field of Protein Drugs for Intracellular Targets (PDIT) is arelatively novel field, derived, in part, from the completion of theHuman Genome Sequencing Project, which allows identification of a hugenumber of novel intracellular targets for potential medicalinterventions, through administration of protein drugs, gene silencing,RNA or DNA editing, or protein replacement therapy. Conceptually, suchtherapeutic strategies can be useful for treatment of almost any medicaldisorder. Specific, highly attractive candidate proteins within the PDITfield are the CRISPR (clustered regularly interspaced short palindromicrepeats)-related proteins, and specifically, the Cas9 Protein. Thisrecently-discovered protein is initially a bacterial protein,naturally-used by bacteria as for an anti-viral agent. Practically, Cas9can be loaded by any RNA sequence, entailing specificity in directingthe protein specifically to any locus within the genome,rationally-selected according to its potential relation to a mutated,defective gene. Cas9 then induces an accurate double-strand cut of theDNA. Naturally-occurring DNA repair mechanisms may then be subsequentlyrecruited, to repair said DNA locus within the malfunctioning gene.Therefore, Cas9 and related proteins enable highly effective geneediting (adding, disrupting or changing the sequence of specific genes)and gene regulation and repair, applicable to species throughout thetree of life. By delivering Cas9 protein and an appropriate guide RNAinto a cell, the organism's genome can therefore be cut at any desiredlocation, and be subjected to editing and repair.

As exemplified below (Example 4), an embodiment of the inventionincludes one or more “molecular nanomotors (MNMs)” linked to the Cas9protein, having a potential role in DNA or RNA editing.

Another embodiment of the invention, relates to a therapeutic protein,administered as a replacement therapy. Such replacement therapy may beneeded in the treatment of a disease, associated with reduced levels ofa physiologically-important protein, due to its deficiency or mutations.In such case, the respective protein may be delivered exogenously, as adrug. Since protein is a charged macro-molecule, many times it isincapable of trans-membrane delivery, unless conjugated to a deliverysystem such as the MNMs of the invention.

MNMs according to embodiments of the invention are typically hydrophobic[typically, without limitation, having octanol to water partitionco-efficient (log P)>1], dipolar, uncharged chemical moieties, designedaccording to the principle of asymmetrical polarity (explained above).As discussed, this unique set of features of the MNM (namely, beinghydrophobic, of overall neutral charge, but being polar, with focusedpartial negative charges and dispersed partial positive charges, createsa unique vectorial system when put in the internal membrane electricfield, entailing movement of the molecule within the phospholipidmilieu, from the membrane/water interface to the membrane center. Whenattached to a drug, this molecule respectively pulls the drug to themembrane core.

As schematically illustrated in FIG. 1B, Conjugates according toembodiments of the invention typically include “Molecular NanoMotor(s)(MNMs)” as described above, being an E, E′ or E″ moiety [demonstrated,for example, by the moiety according to Formula (X)]. The “MolecularNanoMotor (MNM)” is a combination of the following structural elements:

(i). A negative pole (group A of moiety E, E′ or E″), typicallycomprising at least one electronegative atom(s), selected from a halogen[for example, fluorine atom(s)] or oxygen, arranged in space as afocused, spherical (or near spherical) arrangement. Due to theelectron-withdrawing properties of such atoms, and their structuralarrangement in space, the negative pole of the Conjugate is anelectron-rich focus.

(ii). A positive pole (group B of moiety E, E′ or E″), comprisingrelatively electropositive atoms, selected from carbon, silicon, boron,phosphor and sulfur, arranged to enable maximal interaction withadjacent hydrocarbon chains, when put in a phospholipid membrane,preferably through arrangement as an aliphatic or aromatic structure oflinear, branched or cyclic chains, or combinations thereof. In anembodiment of the invention, the positive pole comprises linear,saturated hydrocarbon chain(s), or a steroid moiety, such ascholesterol, bile acids, estradiol, estriol, or derivatives orcombinations thereof. Optionally, the Conjugate of the invention maycomprise several negative pole and several positive pole structuralmotifs, for example, sequentially-arranged perfluro- and oxygen-motifs,separated by hydrocarbon chains, exemplified in the Compound accordingto Formula VIIIa.

In addition to the “Molecular NanoMotor(s) (MNMs)”, a Conjugateaccording to embodiments of the invention may also comprise one or morelinkers (L) and cleavable groups (Q), as further described above. Thelinkage of a drug D to the molecular nanomotor(s) E, E′ or E″ can beeither directly, or through moiety L or Q; said linkage can be throughcovalent or non-covalent bonds, such as electrostatic or coordinativebonds.

Embodiments of the invention further relate to the use of Conjugatesaccording to the invention, comprising therapeutically-useful drugs,such as proteins or oligonucleotides (e.g., siRNA or ASO), for thetreatment of medical disorders in a subject in need thereof. The medicaldisorders may be, without being limited, degenerative disorders, cancer,traumatic, toxic or ischemic insults, infections or immune-mediateddisorders, in which specific protein(s) play(s) a role in either diseaseetiology or pathogenesis, and where modulation of the expression of therespective gene(s), through siRNA or antisense mechanisms, or modulationof the activity of the respective protein by a therapeutic protein or byprotein replacement therapy, may have beneficial effects in inhibitingdisease-related processes or treating the underlying disease.

For example, Conjugates according to embodiments of the invention, maybe used as antisense therapy, which is a form of medical treatmentcomprising the administration of a single-stranded or a double-strandednucleic acid strands (DNA, RNA or a chemical analogue), that binds to aDNA sequence encoding for a specific protein, or to the respectivemessenger RNA (mRNA) where the translation into protein takes place.This treatment may act to inhibit the expression of the respective gene,thereby preventing the production of the respective protein.Alternatively, the Conjugates of the invention may comprise therapeuticproteins, such as the Cas9 protein.

The terms “drug” or “medicament” in the context of the present inventionrelate to a chemical substance, that when administered to a patientsuffering from a disease, is capable of exerting beneficial effects onthe patient. The beneficial effects can be amelioration of symptoms, orcounteracting the effect of an agent or a substance, that play(s) a rolein the disease process. The drug may comprise a small molecule or amacromolecule, such as, a protein, or single- or double-stranded RNA orDNA, administered to inhibit gene expression. Among others, the drug maycomprise siRNA or ASO. In some embodiments, the drug is aimed attreating degenerative disorders, cancer, ischemic, infectious, toxicinsults, or immune-mediated disorders.

The term “biological membrane” according to the invention, refers to anyphospholipid membrane related to a biological system. Examples for suchphospholipid membranes are the plasma membrane of cells, intercellularmembranes, or biological barriers, such as, the blood-brain-barrier(BBB), the ocular-blood-barrier (BOB), or the placenta barrier.

Embodiments of the invention provide Conjugates, comprising MNMsaccording to embodiments of the invention, and a drug. Embodiments ofthe invention further provide pharmaceutical compositions, comprisingthe Conjugates described herein, and pharmaceutically-acceptablecarrier(s) or salt(s).

Other embodiments of the invention, describe methods for treatment ofmedical disorders, comprising administration to a patient in need,pharmaceutical composition of the Conjugates of the invention.

In some embodiments, the medical disorder is cancer. In some specificembodiments, the cancer is, among others melanoma or uterine cervicalcancer.

According to some embodiments, the Conjugates and pharmaceuticalcompositions of the invention may be used to achieve efficient deliveryand effective performance of a replacement protein therapy or genetherapy [for example, without limitation siRNA or antisense therapy(ASO)] in vivo, in the clinical setting.

A Conjugate according to embodiments of the invention may beadvantageous in improving delivery of siRNA, ASO or a therapeuticprotein through cell membranes or through biological barriers, such asthe Blood-Brain-Barrier (BBB), thus improving the performance of saidmacromolecule drug in one or more aspects, such as, for example,efficacy, toxicity, or pharmacokinetics.

As described above in a non-limiting potential Mechanism Of Action(MOA), Conjugates according to embodiments of the invention, comprisinga drug such as siRNA or a therapeutic protein, conjugated to MNM(s),undergo trans-membrane delivery when interacting with a phospholipidmembrane. This mechanism of action is schematically summarized in FIG.2. Due to the principle of asymmetrical polarity, described in FIGS. 1aand 2, initially, the MNMs move from the membrane surface to themembrane core, energized by the internal membrane electric field (i). Asthe second stage [FIG. 2 (ii)], the macromolecule, linked to the MNMs,is forced to approach the membrane surface, thus perturbing thehydration shells of both the cargo macromolecule drug and thephospholipid head-groups. Consequently, there is lateral movement of thephospholipid head-groups, and formation of transient membrane pores,through which the macromolecule drug is delivered into the cell.Subsequent closure of the transient pore then takes place with membranehealing [FIG. 2 (iii)].

In an example, schematically presented in FIG. 1B, the Conjugate of theinvention comprises a cargo drug (moiety D), being siRNA, ASO or atherapeutic protein, and a disulfide group, for entrapment of the cargodrug in the cytoplasm, due to the ambient reductive environment.

In another embodiment of the invention, entrapment of siRNA in thecytoplasm may be achieved through the administration of a Conjugate,where D is a double-stranded RNA, which is a Dicer substrate, namely,comprising 23-30 nucleotides, selected according to the genetic codesuitable for silencing a specific target gene. One or several MNMs maythen be linked to such oligonucleotide drug. Preferably, MNMs areattached at the 3′-end and/or the 5′-end of the sense (“passenger”)strand, and/or at the 5′-end of the antisense (“guide”) strand. Uponadministration of the Conjugate, the MNMs will enable the trans-membranedelivery of the macro-molecule drug. Subsequent cleavage of the dsRNA bythe Dicer enzyme in the cytoplasm will then remove the MNM(s) at the3′-end of the passenger strand, and/or at the 5′-end of the guide stand,thus releasing the siRNA. The siRNA, due to its numerous negativecharges, is eventually entrapped in the cytoplasm, where it interactswith the RISC complex, resulting in silencing of the target gene. This,Dicer-mediated mechanism of intracellular entrapment is schematicallyillustrated in FIG. 3.

In yet another mechanism, entrapment of siRNA or ASO within thecytoplasm can be achieved in the case that E, E′ or E″ comprises a Qmoiety, being a chelator for calcium ion(s), bound via coordinativebonds to phosphate groups of the oligonucleotide drug. Such binding canbe mediated, for example, by Ca⁺² ions. Such Conjugatse may be stable inthe plasma, due to the relatively high ambient Ca⁺² levels (about 1 mM).Moreover, due to the MNMs, the Conjugates will manifest trans-membranedelivery into the cells. Once inside the cytoplasm, the lowcytoplasmatic Ca⁺² levels will induce de-complexation, releasing thecargo oligonucleotide, which will then interact with its target sites,such as the Dicer or the RISC complex, for gene silencing.

Conjugates according to embodiments of the invention have the structure,as set forth in general Formula (I):

including pharmaceutically acceptable salts, hydrates, solvates andmetal chelates of the compound represented by the structure as set forthin Formula (I), and solvates and hydrates of the salts, wherein:

-   D is a drug to be delivered across biological membranes. D may be a    small-molecule drug, a peptide, a protein, or a native or modified,    single-stranded or double-stranded DNA or RNA, such as siRNA or ASO;-   y, z and w are each an integer, independently selected from 0, 1, 2,    3, 4, 5, 6; at least one of y, z or w is different from 0. In one    embodiment, y=1, z=o and w=0; in another embodiment y=1, z=1 and    w=0.    E, E′, or E″ can be the same or different, each having the structure    as set forth in general Formula (II):    (A)_(a)-B-L₁-Q₁-L₂-Q₂-L₃   Formula (II)    wherein-   B (a positive pole as described above) is selected from the group    consisting of, linear, cyclic or branched C₁, C₂, C₃, C₄, C₅, C₆,    C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, alkyl or hetero-alkyl;    -   linear, cyclic or branched C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀,        C₁₁, C₁₂, C₁₃, C₁₄ alkylene or heteroalkylene;    -   C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄ aryl or heteroaryl;    -   one or more steroid moiety (such as, cholesterol, bile acid,        estrogen, estradiol, estriol), nucleoside, nucleotide; and any        combination thereof;    -   wherein each group is optionally substituted by hydroxyl, amine,        or thiol;-   Q₁ and Q₂ are each independently an optionally cleavable group,    selected from null, ester, thio-ester, amide [e.g., —C(═O)—NH— or    —NH—C(═O)—], carbamate [e.g., —O—C(═O)—NH— or —NH—C(═O)—O—], urea    [—NH—C(═O)—NH—], disulfide [—(S—S)—], ether [—O—], imidazole,    triazole, a pH-sensitive moiety, a redox-sensitive moiety; a metal    chelator, including its chelated metal ion; and any combinations    thereof;-   L₁, L₂ and L₃ are each independently selected from null and the    group consisting of:    -   linear, cyclic or branched C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉,        C₁₀, C₁₁, C₁₂, C₁₃ or C₁₄, alkyl or hetero-alkyl;    -   linear, cyclic or branched C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀,        C₁₁, C₁₂, C₁₃ or C₁₄ alkylene or heteroalkylene;    -   C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃ or C₁₄ aryl or        heteroaryl;    -   —(O—CH₂—CH₂)_(u)—, wherein u is an integer of 1, 2, 3, 4, 5;    -   nucleoside, nucleotide; imidazole, azide, acetylene; and any        combinations thereof;    -   wherein each group is optionally substituted by one or more        hydroxyl, amine, or thiol;    -   wherein each of Q₁, Q₂, L₁, L₂ and L₃ optionally comprises a T        moiety; wherein T is an initiator group, selected from C₄, C₅,        C₆-1,2-dithiocycloalkyl(1,2-dithiocyclo-butane;        1,2-dithiocyclo-pentane; 1,2-dithiocyclohexane;        1,2-dithiocycloheptane); γ-Lactam (5 atoms amide ring), δ-Lactam        (6 atoms amide ring) or ε-Lactam (7 atoms amide ring);        γ-butyrolactone (5 atoms ester ring), δ-valerolactone (6 atoms        ester ring) or ε-caprolactone (7 atoms ester ring); and T        optionally comprises an amine group.-   A is selected from the structures as set forth in Formulae (III),    (IV), (V) and (VI) (a negative pole as described above):

-   -   M is selected from —O— or —CH₂—; and g, h and k are each        individually an integer selected from the group consisting of 0,        1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16; * is        —H, or a point of linkage to B; a is an integer, selected from        1, 2, 3 or 4.

The linkage of D to other moieties of the molecule can be throughcovalent, electrostatic, or coordinative bonds. In the case that thebond is covalent, linkage can be through a Q₁ or Q₂ group, selected fromthe group consisting of ether, ester, amide, thioester, thioether andcarbamate groups. In the case that the bond is coordinative, it involvesa Q₁ or Q₂ group that is a metal chelator, and the linkage preferablyinvolves coordination of calcium ion(s). An example for electrostaticlinkage, can be a salt bridge between amine groups of moiety L₁, L₂ orL₃ of E, E′ or E″, and negatively-charged phosphate groups of D. In casethat D is an oligonucleotide, linkage can be to the nucleobase, to theribose moiety (e.g., through the 2′, 3′ or 5′ positions of the ribose),or to the phosphate moiety of the nucleotide; linkage can be either to aterminal, or to a non-terminal nucleotide of the oligonucleotide chain;linkage can be through a natural or through a modified nucleotide. Inthe case that D is a protein, its linkage to the other moieties of themolecule can be through linkage to side chain(s) of the protein's aminoacids, such as lysine, cysteine, glutamate or aspartate.

The term “oligonucleotide”, in the context of the invention, may includeDNA or RNA molecules, each being a single-stranded or double-strandedsequence of one or more nucleotides. Each nucleotide comprises anitrogenous base (nucleobase), a five-carbon sugar (ribose ordeoxyribose), and a phosphate group. The nucleobases are selected frompurines (adenine, guanine) and pyrimidines (thymine, cytosine, uracil).In addition, the term may also refer to modified forms of nucleotides,where the modification may be at the backbone of the molecule (e.g.,phosphorothioate, peptide nucleic acid) or at the nucleobase (e.g.,methylation at the 2′ position of the ribose group in RNA, or attachmentof fluorine atoms at that site). These modifications may enableproperties such as improved stability or improved pharmacokinetics ofthe oligonucleotide in body fluids. The use of such modifiedoligonucleotides is therefore also within the scope of the invention.

In one embodiment, a method for specific inhibition of gene expressionis disclosed, applicable either in vitro or in vivo. The methodcomprises the utilization of a Conjugate of the invention or apharmaceutical composition comprising the Conjugate, where D is siRNA orASO, designed to silence the expression of a specific gene, whichencodes for a pathogenic protein, that has a role in the etiology orpathogenesis of disease.

Accordingly, Conjugates according to embodiments of the invention may beused for the treatment of a medical disorder. Embodiments of theinvention therefore disclose a method for medical treatment, comprisingthe administration to a patient in need, therapeutically effectiveamounts of a pharmaceutical composition according to embodiments of theinvention. In one embodiment, the administered pharmaceuticalcomposition may comprise siRNA or an antisense oligonucleotide, activein inhibiting the expression of a specific gene encoding for adisease-related protein.

In one embodiment of the invention, there is provided a Conjugateaccording to general Formula (I), comprising MNMs, being an E, E′ or E″moiety, having the structure as set forth in Formula (VII):

where m is an integer, selected from the group consisting of null and 1,2, 3, 4, 5, 6, 7, 8, 9 or 10; k is an integer, selected from 0, 1, 2, 3or 4; U is selected from the group consisting of null, —O—, N and NH;and the E, E′ or E″ moiety is linked to D; including pharmaceuticallyacceptable salts, hydrates, solvates and metal chelates of the Compoundrepresented by the structure as set forth in Formula (VII), and solvatesand hydrates of the salts.

In one embodiment, k=1.

In another embodiment, the steroid moiety is substituted by residue oflithocholic acid, or a related analogue.

In another embodiment, L₁, L₂ and L₃ are each individually selected formnull and a linear, cyclic or branched C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈hydrocarbon chain; L₁, L₂ and L₃ can be the same or different.

In another embodiment Q₁ or Q₂ is an amide group, an ester, a carbamate,or a disulfide.

In still another embodiment, Q₁ or Q₂ is imidazole.

In still another embodiment, L₁, L₂ or L₃ comprises a T moiety, being1,2-dithiocyclo-butane, optionally linked to an amide or amine group.

In a more specific embodiment, there is provided a Conjugate accordingto general Formula (I), which includes E, E′ or E″, having the structureas set forth in Formulae (VIIa):

including pharmaceutically acceptable salts, hydrates, solvates andmetal chelates of the Compound represented by the structure as set forthin Formula (VIIa), and solvates and hydrates of the salts; wherein gstands for an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14.

In a preferred embodiment, g=2 or g=12.

In another embodiment, there is provided a molecule according to generalFormula (I), which includes E, E′ or E″, having the structure as setforth in Formulae (VIII):

including pharmaceutically acceptable salts, hydrates, solvates andmetal chelates of the compound represented by the structure as set forthin Formulae (VIII) and solvates and hydrates of the salts; where kstands for an integer, selected from the group consisting of 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16; h stands for an integer,selected from the group consisting of 0, 1, 2, 3, 4; U is selected fromthe group consisting of null, and a C₁, C₂, C₃ or C₄ alkyl; Z isselected from hydrogen, hydroxyl and amine groups; Y is selected from—CH— and a nitrogen atom.

In preferred embodiments, k=1, and h=1.

In another embodiment, there is provided a molecule according to generalFormula (I), which includes E, E′ or E″, having the structure as setforth in Formulae (IX):

including pharmaceutically acceptable salts, hydrates, solvates andmetal chelates of the compound represented by the structure as set forthin Formulae (IX) and solvates and hydrates of the salts.

In another embodiment, there is provided a molecule according to generalFormula (I), which includes E, E′ or E″, having the structure as setforth in Formula (X):

including pharmaceutically acceptable salts, hydrates, solvates andmetal chelates of the compound represented by the structure as set forthin Formulae (X) and solvates and hydrates of the salts; wherein w standsfor an integer of 0, 1, 2 or 3; t stands for an integer of 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14; p stands for an integer of 0, 1,2 or 3; R₁ stands for hydrogen, or an alkyl group of 1, 2 or 3 carbonatoms.

In another embodiment, there is provided a molecule according to generalFormula (I) and Formula (XI), which includes E, E′ or E″, having thestructure as set forth in Formulae (XI):

including pharmaceutically acceptable salts, hydrates, solvates andmetal chelates of the compound represented by the structure as set forthin Formulae (XI) and solvates and hydrates of the salts.

In another embodiment, there is provided a molecule according to generalFormula (I) and Formula (VIII), which includes E, E′ or E″, having thestructure as set forth in Formulae (XII):

including pharmaceutically acceptable salts, hydrates, solvates andmetal chelates of the compound represented by the structure as set forthin Formulae (XII) and solvates and hydrates of the salts; wherein bstrands for an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; J is null,nitrogen or an oxygen atom.

In a specific embodiment, there is provided a molecule according togeneral Formula (I) and Formula (XII), which includes E, E′ or E″,having the structure as set forth in Formulae (XIII):

In another specific embodiment, there is provided a Conjugate accordingto general Formula (I) and Formula (XII), which includes E, E′ or E″,having the structure as set forth in Formulae (XIV):

In another embodiment, the invention concerns a Conjugate, having thestructure as set forth in general Formula (I), which comprises E, E′ orE″, having the structure as set forth in Formula (XVI):

including pharmaceutically acceptable salts, hydrates, solvates andmetal chelates of the compound represented by the structure as set forthin Formulae (XV) and solvates and hydrates of the salts; where y standsfor an integer, selected from the group consisting of 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11 or 12. In a preferred embodiment, y=3.

In another embodiment, the invention concerns a Conjugate, having thestructure as set forth in general Formula (I), which comprises E, E′ orE″, having the structure as set forth in Formula (XVI):

including pharmaceutically acceptable salts, hydrates, solvates andmetal chelates of the compound represented by the structure as set forthin Formulae (XVI) and solvates and hydrates of the salts; where f standsfor an integer, selected from the group consisting of 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16.

Also within the scope of the invention are molecules termed“precursors”. A “precursor” in the context of the invention, is achemical moiety, used in the synthesis of Conjugates according toembodiments of the invention. Often, the precursor comprises chemicalgroups, destined to be removed during the synthesis of the Conjugate, instages such as attachment of a therapeutic molecule or a macromoleculeto the MNMs of the invention.

In one embodiment, the precursor has the structure, as set forth inFormula (XVII):

wherein W is a moiety, selected from E, E′ or E″, as described in to anyof Formulae II, VII, VIIa, VIII, IX, X, XI, XII, XIII, XIV, XV or XVI.This precursor is useful, without limitation, for attachment to the5′-end of an oligonucleotide.

Another precursor of the invention has the structure according toFormula (XVIII):

wherein G is a moiety, selected from E, E′ or E″ as described in any ofFormulae II, VII, VIIa, VIII, IX, X, XI, XII, XIII, XIV, XV or XVI. Thisprecursor may be useful, among others, for attachment to the 3′-end ofan oligonucleotide; DMT=Dimethoxytrityl; CPG=Controlled Pore Glass(CPG).

In other embodiments of the Invention, the precursor has the structureas set forth in any of Formulae II, VII, VIIa, VIII, IX, X, XI, XII,XIII, XIV, XV or XVI, wherein at the point of linkage to D, there islinkage to a group selected from phosphoroamidite, an activated ester,azide or acetylene. The latter two groups may be useful for attachmentto D by “click chemistry”, for example without limitation, through theAzide-alkyne Huisgen cyclo-addition reaction.

Embodiments of the invention may further include pharmaceuticalcompositions, comprising a Conjugate, comprising a molecule according toany of Formulae II, VII, VIIa, VIII, IX, X, XI, XII, XIII, XIV, XV orXVI, and a pharmaceutically-acceptable salt or carrier.

The invention also comprises methods for specific inhibition of geneexpression, in vitro or in vivo. In one embodiment, the method mayinclude utilization of a Conjugate according to any of Formulae II, VII,VIIa, VIII, IX, X, XI, XII, XIII, XIV, XV or XVI; or a respectivepharmaceutical composition, where D is siRNA or an ASO, designed tosilence the expression of a specific gene. In some embodiments, the geneencodes for a pathogenic protein, having a role in the etiology orpathogenesis of a disease. In some embodiments, D is a therapeuticprotein.

Conjugates according to embodiments of the invention may be used for thetreatment of a medical disorder. Embodiments of the invention includemethods for medical treatment, comprising the administration to apatient in need therapeutically effective amounts of a pharmaceuticalcomposition, comprising a Conjugate according to any of Formulae II,VII, VIIa, VIII, IX, X, XI, XII, XIII, XIV, XV or XVI; where D is a druguseful for treatment of the respective medical disorder.

In one embodiment, the method is for genetic treatment with siRNA orASO, said method comprising the administration to a patient in needtherapeutically effective amounts of a pharmaceutical composition,comprising a Conjugate of the invention according to any of Formulae II,VII, VIIa, VIII, IX, X, XI, XII, XIII, XIV, XV or XVI; where D is siRNA,an ASO or a therapeutic protein, useful in inhibiting the expression ofa gene which plays a role in the disease of the specific patient.

In another embodiment of the invention, the invention includes a methodfor medical treatment of a disease by therapeutic a protein, where D isa protein to be delivered across biological phospholipid membranes intocells, or through biological barriers, such as the blood-brain barrier.Said cells are either in cell culture in vitro, or in a living animal ora human subject in vivo.

In some embodiments, the cell is a neoplastic cell. In some embodiments,the neoplastic cell is a tumor cell. In some embodiments, the neoplasticcell is a cell within a metastasis. The cell may be a eukaryotic cell, aeukaryotic cell infected with an oncogenic agent, a human cell, a cellthat is a pre-cancerous cell, or any combination thereof. The cell maybe a cell within a cell culture, or within a living animal or a humansubject.

In yet another embodiment of the invention, D is a protein, administeredas a replacement therapy, e.g., to replace a mutated, malfunctioningprotein, thus addressing a physiological need. In another embodiment, Dis a protein that has as role in gene regulation, including, amongothers, proteins that have a role in DNA or RNA editing (adding,disrupting or changing the sequence of specific genes).

In one embodiment, said protein may be a member of the CRISPRs(clustered regularly interspaced short palindromic repeats) relatedproteins. Specifically, said protein can be, or may comprise the Cas9protein (CRISPR associated protein 9), an RNA-guided DNA nucleaseenzyme, or an analogue thereof.

In one of the embodiments of the invention, it describes a method forgenetic treatment of a medical disorder, said method comprisingadministration to a patient in need, therapeutically effective amountsof a pharmaceutical composition, comprising a conjugate according toFormula (I), where D is a CRISPR protein, such as Cas9, administeredtogether with an appropriate guide oligonucleotide, thus achievingdelivery of the protein, loaded with a respective guide oligonucleotide,into the cells, where the CRISPR protein can exert its genome editingactivity. A guide oligonucleotide, in this context, is a sequence of RNAor DNA that guides the Cas9 protein to a specific locus (place) on theDNA, in order to induce a double-strand DNA cleavage at that site, thusenabling to repair a local defect in the genetic material. In the caseof Cas9, the guide oligonucleotide is short segment of RNA, the sequenceof which is complementary to the sequence of the target DNA locus.

Therefore, conjugates according to embodiments of the invention, and therespective pharmaceutical compositions and methods may be beneficial,among others, in the treatment of medical disorders, selected, amongothers, from cancer, toxic insults, ischemic disease, infectiousdisease, protein storage disease, trauma, immune-mediated disease, or adegenerative disease.

According to some embodiments, the medical disorder is cancer. As usedherein, the term “cancer” refers to the presence of cells possessingcharacteristics, typical of cancer-causing cells, such as uncontrolledproliferation, loss of specialized functions, immortality, significantmetastatic potential, significant increase in anti-apoptotic activity,rapid growth and proliferation rate, and certain characteristicmorphology and cellular markers. Typically, cancer cells are in the formof a tumor, existing locally within an animal, or circulating in thebloodstream as independent cells, as are, for example, leukemic cells.

In the field of neurological disorders, conjugates according toembodiments of the invention may be useful, among others, in thetreatment of neurodegenerative disorders, such as Alzheimer's disease,Motor Neuron Disease, Parkinson's disease, Huntington's disease,multiple sclerosis and Creutzfeldt-Jacob disease.

EXAMPLES

Some examples will now be described, in order to further illustrate theinvention, and in order to demonstrate how embodiments of the inventionmay be carried-out in practice.

In the following Examples, described are Conjugates, comprising the MNMsof the invention, attached to a single-stranded or to a double-strandedoligonucleotide. These Examples demonstrate the entire spectrum of theinvention, namely, that the MNM of the Invention is: (i). Successfullysynthesized; (ii). Successfully conjugated to a macromolecule drug(e.g., single-stranded or double-stranded DNA or RNA); (iii). Enableefficient delivery of heavily-charged macro-molecules (e.g., carrying 29or 58 negative charges) across hydrophobic phospholipid membranes intocells; and (iv). Enable these macro-molecules, once inside the cells, toexert a useful biological activity (e.g., gene silencing).

Example 1 A General Method for Synthesis of Conjugates According toEmbodiments of the Invention, Comprising Oligonucleotides

Initially, a gene to be silenced is chosen based on its role in diseaseetiology or pathogenesis. Then, based on bio-informatic methodologiesknown in the art, the nucleotide sequence (typically 19-21 base-pairsdouble-stranded RNA for a RISC substrate, or 25-29 base-pairsdouble-stranded RNA for a Dicer substrate) is determined.

Synthesis is carried out in the 3′ to 5′ direction. Solid phasesynthesis is applied, using phosphoramidite building blocks, derivedfrom protected 2′-deoxynucleosides (dA, dC, dG, and T), ribonucleosides(A, C, G, and U), or chemically modified nucleosides, e.g. LNA (lockednucleic acids) or BNA (bridged nucleic acids). The building blocks aresequentially coupled to the growing oligonucleotide chain, in the orderdetermined by the sequence of the desired siRNA.

Following the construction of the oligonucleotide, an E moiety of theinvention is added, as one of the building blocks of theoligonucleotide. The E moiety is added as its precursor form, asdescribed above. For linking the compound to the 5′-end of theoligonucleotide, a precursor according to Formula (XVII), comprising aphosphoramidite moiety is utilized. For linking the compound at the3′-end of the oligonucleotide, a precursor according to Formula (XVIII)is utilized. Among others, this precursor may comprise acetylene orazide moieties to mediate linkage of the E moiety to the oligonucleotidechain. The process is fully automated. Upon completion of the assemblyof the chain, the product is released from the solid support intosolution, de-protected, and collected. The desired Conjugate is thenisolated by high-performance liquid chromatography (HPLC), to obtain thedesired conjugated oligonucleotide in high purity. In the case of siRNA,each of a complementary RNA strands is synthesized separately, and thenannealing of the two strands is performed in standard conditions knownin the art, to yield the desired double-stranded siRNA.

Example 2 Chemical Synthesis of E Moieties of the Invention (E, E′ orE″)

Molecular design is performed by Aposense, Ltd. Petach-Tiqva, Israel,and synthesis is performed by Syncom BV, the Netherlands. The startingmaterial perfluoro-tertbutanol is commercially-available. In thisexample, the E moieties are designed to be linked to the 5′-end of theoligonucleotide, and therefore a phosphoramidite moiety is added at thelast step of the synthesis, towards conjugation to the oligonucleotidechain.

Example 2a A Method for Synthesis of an E Moiety According to Formula(VIIa)

The synthesis starts from estradiol, an estrogen that iscommercially-available.

Synthesis is performed according to Scheme 1. For example, estradiol wasprotected by a benzyl group to provide compound 11. Allylation ofalcohol 11 (25.6 g) under optimized reactions conditions (allyl bromide,NaH, cat. TBAI, THF, reflux, 16 h) afforded allyl ether 24 (21.85 g,77%) as a white solid (purified by successive trituration in heptane andMeOH). Regio-selective hydroboration of the terminal alkene 24 (21.8 g)with 9-BBN, upon standard oxidative workup (NaOH/H₂O₂) provided alcohol22. Mitsunobu reaction of the alcohol 22 (13.6 g) with excessperfluoro-tert-butanol under optimized reaction conditions (DIAD, PPh₃,4 A MS, THF, RT, 16 h) afforded the desired ether 21. Compound 21 wassubjected to catalytic hydrogenation (10% Pd/C, RT) using a mixture(1:1) of THF and 2,2,2-trifluoroethanol as solvent (5 bars, Parrreactor) to afford (after ˜18 h) the phenol 26 as off-white solid.De-benzylation was then performed, followed by alkylation, using aTHP-protected bromobutanol. The protecting group was then removed,followed by attachment of the phosphoramidite group, as the last step tothe desired compound. This Product was then subjected to conjugation tothe oligonucleotide chain, via the phosphoramidite group, as the finalbuilding block of synthesis of the oligonucleotide chain, at the 5′-end.

Example 2b A Method for Synthesis of the E Moiety According to Formula(XVI)

The synthesis starts with lithocholic acid, a bile acid that iscommercially-available. The synthesis follows synthetic Scheme 2:

For example, 25 g of material 1 were converted to correspondingmethyl-ester in a quantitative yield. 25 g of material 2 were reactedwith TBDMSCl NS 29 g (87%, NMR). Pure compound 3 was obtained. Reductionof compound 3 (29 g) to 4 with NaBH₄ THF/MeOH gave, after work up andpurification, compound 4 (85%) by NMR, still with some traces ofcompound 3. Mitsunobu reaction of material 4 with perfluoro t-butanolgave, after work-up column chromatography and trituration from MeOH,33.5 g (92%) of compound 5, which was de-protected thereafter, to givesteroid 6. Steroid 6 (2.5 g) was then coupled to THP-protectedbromotetradecanol. The coupling took 3 days, and 4 equivalents ofTHP-protected bromotetradecanol were needed to reach completeconversion. The product was purified by column chromatography. Afterremoval of the protecting group (THP) with MeOH/1,4-dioxane (HCl, 4N)/THF, product 7 was purified by column chromatography to removeimpurities. Product 7 (1.5 g, c.y. 48%) was obtained as white solid.Product 7 was then converted into the desired compound 8, by attachmentof the phosphoramidite group. This Product was then subjected toattachment to the oligonucleotide chain, as the final building block ofsynthesis of the oligonucleotide chain, at the 5′-end.

Example 2c A Method for Synthesis of the E Moiety According to Formula(IX)

Intermediate 26 is synthesized as described in Example 2a. Then thesynthesis is performed according to the following Scheme 3.

For example, dithiol-butyl amine (0.5 g) with iodine under basicconditions afforded the 1,2-dithiane 10 (3.13 g, 90%) as acrystalline-white solid. The alcohol corresponding to intermediate 11 iscommercially-available, and was protected with dimethoxytrityl (DMT).Reductive amination with amine 10 (258 mg) in presence of NaBH(OAc)₃afforded the desired secondary amine 4 (330 mg, 91%) as major product.Intermediate 26 is then attached to intermediate 4 throughcarbmoylation, as known in the art. DMT is then removed, and aphosphoramidite group is attached, to yield a precursor compound. Thisprecursor is then subjected to conjugation to the oligonucleotide chain,as the final building block of the chain, at the 5′-end. Linkage isperformed through an oxygen atom. Said conjugation yields the desiredConjugate, comprising an E moiety according to Formula (IX).

Example 3 Examples of Conjugation of MNMs to Oligonucleotide Chains

Examples of structures of precursors; and respective compounds, whenconjugated to an oligonucleotide chain.

a. 5′ Modification:

Precursor:

As attached to an oligonucleotide:

b. 3′ Modification:Precursor:

wherein DMT=Dimethoxytrityl; and CPG=Controlled Pore Glass (CPG) as asolid support for the synthesis of the oligonucleotide.

As attached to an oligonucleotide:

c. 5′ Internal Modification:

In this modification, E comprises a nucleotide (e.g., thymine): Thismodification can serve for attachment of an E moiety within anoligonucleotide chain, rather than at a terminal position.

Example 4 An Exemplary Structure of a Conjugate of the Invention,Comprising a Protein (for Example, without Limitation Cas9), Conjugatedto E Moieties of the Invention

A structure of an MNM of the invention, conjugated to the Cas9 proteinis schematically illustrated in FIG. 4. MNMs E, E′ or E″ according toembodiments of the invention are attached through a linker group to theprotein. Binding is performed through carbamate or amide bonds to lysineside-chains on the protein surface. For attachment, active esters areused. For this purpose, the alcohol is converted to an active ester(e.g., N-hydroxysuccinimide, NHS), that preferentially reacts withnitrogen of the protein lysine side-chains over oxygen (water). Reactionis performed according to the following Scheme:

Possible derivatizing agents are:

-   -   a) Phosgene: linkage is through chloroformate ester.    -   b) Disuccinimidyl carbonate (X═N-hydroxysuccinimide): linkage is        through a succinimidyl carbonate.    -   c) Carbonyldiimidazole (CDI, X=Imidazole): linkage is through        imidazolyl carbamate.

Protein labeling with any of these groups takes place in an amine-free(not Tris), slightly basic buffer (pH=8-9). The linkage point ishydrophobic, thus requiring a co-solvent (normally DMF or DMSO) for thereaction with proteins to take place. High reactivity means, on the onehand shorter reaction times, but, on the other hand also lower nitrogenover oxygen selectivity, and shorter lifetime in aqueous buffer. Whenthe product is a carbamate, it may be susceptible to enzymatic cleavage.Of the three options above, carbonyl-di-imidazole has the highestnitrogen over oxygen selectivity, as well as the simplest synthesis, andis therefore preferred. On the other hand, carbonyl-di-imidazole isassociated with a longer protein derivatization time (probablyovernight). The number of E, E′ or E″ moieties per protein molecule isdetermined by pre-setting of the desired molar ratios.

Example 5 Cellular Uptake of Conjugates, Comprising DNAOligonucleotides, Conjugated to One or Two Molecular NanoMotors of theInvention

In the following Examples, cellular uptake of Conjugates, comprisingApo-Si MNMs according to Formula (XI) or Formula (XVI) is provided,attached to either Cy3-labeled single-stranded 29-mer DNA sequence(carrying 29 negative charges), or to a double-stranded 58-mer DNAsequence (carrying 58 negative charges) is described. The sequences ofthe DNA oligonucleotides were 5′Apo-si-TT-iCy3-CGGTGGTGCAGATGAACTTCAGGGTCA (SEQ ID NO: 1) and5′Apo-si-TGACCCTGAAGTTC ATCTGCACCACCGAA (SEQ ID NO: 2). iCy3 means thefluorophore Cy3, at an internal position along the sequence). Thesesequences (synthesized, for example without limitation, by IDT, Iowa,USA) were chosen randomly, aimed at serving as an example for thetrans-membrane delivery into the cells. The incorporation of thefluorophore served as a tool to detect the location of the examinedConjugate. Performance in various cell lines is presented, todemonstrate that the trans-membrane delivery of macromolecules by theApo-Si MNMs is universal, and is not limited to a specific cell type.

Example 5a 3T3 Cells

In order to assess the ability of an MNM of the invention to deliver a29-mer single strand DNA (ssDNA) oligonucleotide into cells, an assay invitro was performed. One day before experiment, NIH-3T3 cells, stablytransfected with the EGFP protein (3T3-EGFP cells) in the exponentialgrowth phase, were plated in 24-well plates, at a density of 4.5×10⁴cells/well with DMEM, plus supplement growth medium (500 μl/well),without antibiotics. Initially, a Cy3-labeled 29-mer ssDNAoligonucleotide, having the sequence of5′Apo-si-TT-iCy3-CGGTGGTGCAGATGAACTTCAGGGTCA (SEQ ID NO: 1). Thissequence was conjugated to a single MNM. The uptake of this Conjugateinto the cells was compared to the uptake of a control compound, beingthe same DNA strand, with Cy3, but without the MNM. The Conjugate wasdiluted in 100 μl/well of Opti-Mem (Life technologies-Cat. 31985062,USA), incubated for 10 minutes in room temperature, and added to thecells at a final concentration of 100 nM. Uptake of the Conjugate by thecells versus Control was evaluated at 8 hours of incubation. At the endof the incubation period, cells were washed with Hank's Buffered SaltSolution (HBSS buffer; Biological Industries, Israel) and subjected toanalysis. Cells were visualized using an Olympus fluorescent microscope(BX51TF; Olympus Optical, U.K.), with UV illumination from a mercurylamp (×20 magnitude). The Cy3-fluorophore was visualized with anexcitation wavelength of 470-495 nm and emission at 590 nm, while theEGFP fluorophore was visualized with excitation at 530-550 nm andemission at 510-550 nm. As shown by fluorescent microcopy in FIG. 5a ,Apo-Si-11, comprising the MNM linked to a 29-mer DNA strand, manifestedefficient delivery across cell membranes into the 3T3-EGFP cells, incontrast to the Control oligonucleotide without the MNM, in which nosignificant uptake was observed.

The ability of the Apo-Si MNM to the deliver 29-mer ssDNAoligonucleotide to 3T3-EGFP cells was also quantified using an ELISAreader (FIG. 5c ). For this purpose, cells at an exponential growthphase were plated one day before the experiment in 24-well plates at adensity of 4.5×10⁴ cells/well with DMEM, plus supplements growth medium(500 μl/well) without antibiotics. Each Cy3-labeled oligonucleotide wasdiluted in 100 μl/well of Opti-Mem), and added to the cells, at a finalconcentration varying from 40 to 100 nM. The accumulation of the Apo-SiMNM-Conjugate within the cells versus the Control Compound without MNMwas evaluated at 24 h of incubation. For this purpose, cells were washedwith HBSS buffer and subjected to analysis. Detection and quantificationof Cy3-positive population was performed using Tecan Infinite® 200 PROmultimode reader (excitation wave length 548±4.5 nm and emission 580±10nm). Uptake of the Apo-Si MNM Conjugate was compared to the uptake ofthe control DNA oligonucleotide at the same concentrations, and resultswere expressed as percentage, compared to Control. As shown in FIG. 5c ,significant uptake of the Conjugate into the cells was observed, ascompared to Control.

Cellular uptake of the Apo-Si MNM, linked to a 29-mer DNAoligonucleotide was also evaluated by flow cytometric analysis (FACS).As described above, one day before the experiment, 3T3-EGFP cells in theexponential growth phase were plated in 6-well plates, at a density of1.5×10⁵ cells/well, with DMEM complete medium, without antibiotics. Eachof the Cy3-labeled oligonucleotides was diluted in 500 μl/well ofOpti-Mem, and added to the cells at a final concentration varying from 1to 40 nM. Delivery of the Conjugate was evaluated at 24-72 h posttransfection. Following the incubation period, cells were trypsinized,supplemented with Hank's Buffered Salt Solution (HBSS buffer; BiologicalIndustries) and centrifuged for 5 min at 1100 rpm. Cells were thenre-suspended with Hank's Buffered Salt Solution, and subjected toanalysis using FACSAria III Cell Sorter (BD Biosciences, San Jose,Calif.), utilizing the Cell Diva software. For each sample, a total of10⁴ events were collected. Detection and quantification of theCy3-positive cell population were performed using measurements of thefluorescence intensity in the cells incubated with the Apo-Si-11Conjugate, relative to that of the cells incubated with the controloligonucleotide, having the same sequence, but devoid of the MNM.

FACS analysis confirmed that Apo-Si MNM is capable of efficient deliveryof a 29-mer ssDNA oligonucleotide to 3T3-EGFP cells. FIG. 5b provides adot plot analysis, showing that in the cell population incubated withthe Apo-Si-11 Conjugate, practically all cells manifested uptake of theConjugate, in contrast to Controls.

We then assessed the ability of Apo-Si-11 to deliver double-strandedoligonucleotide (dsDNA) across the cell membranes. For that purpose, twoApo-Si-11 nanomotors were attached, one at each 5′-end of a 29 bp dsDNAoligonucleotide, labeled by the cy3 fluorophore. Sequence of the dsDNAwas as described above: 5′Apo-si-TT-iCy3-CGGTGGTGCAGATGAACTTCAGGGTCA(SEQ ID NO: 1) and 5Apo-si-TGACCCTGAAGTTCATC TGCACCACCGAA (SEQ ID NO:2). Attachment of the MNM to the oligonucleotide was performed asexemplified in Example 3 above. 3T3-EGFP cells were incubated with 40 nMof the Conjugate, and cellular uptake was evaluated by fluorescentmicroscopy at 24 h of incubation, and was compared to the uptake bycells incubated with a Control identical oligonucleotide, devoid of theMNMs. As described in FIG. 5d , two Apo-Si-11 MNMs were capable ofefficient delivery of the 58-mer dsDNA oligonucleotide into the 3T3-EGFPcells.

This delivery was further demonstrated by FACS. For this purpose,3T3-EGFP cells were plated in 6-well plates, and treated as described inFIG. 5C. Each of the Cy3-labeled oligonucleotide (with and without theMNMs) was diluted in 500 μl/well of Opti-Mem, added to the cells atfinal concentrations of 40 nM, 10 nM and 1 nM. Following a 24 hincubation period, delivery of the oligonucleotides was evaluated byFACSAria III Cell Sorter (BD Biosciences, San Jose, Calif.) and analyzedby Cell Diva software. A total of 10⁴ events were collected for eachsample. Detection and quantification of Cy3-positive population wereperformed using measurements of the fluorescence intensity in the cellsincubated with the Apo-Si MNM Conjugate, relative to that of the cellsexposed to the Control Oligonucleotide devoid of the MNMs. As shown inFIG. 5e , FACS analysis confirmed that two Apo-Si MNMs are capable ofefficient delivery of a 58-mer dsDNA oligonucleotide into 3T3-EGFPcells: (i). Dot plot analysis, showing that only cells incubated withthe Apo-Si-11 Conjugate manifested uptake of the Conjugate, whichaccumulated in practically all cells; (ii). Histogram geomean analysis,indicating a marked signal in the Apo-Si MNM-Conjugate-treated cells, incontrast to low, background levels in cells treated by the Controloligonucleotide devoid of the molecular nanomotors. A cleardose-response was observed in the examined concentrations (40 nM, 10 nM,and 1 nM).

We then used confocal microscopy, in order to further confirm uptake andcytoplasmic localization of the Conjugate, attached to two Apo-Si-11MNMs. Cells were prepared as described above. Nuclear staining with theHoechst 33258 dye (Sigma Aldrich, USA, 1:1000 in HBSS for an hour) wasalso performed. As shown in FIG. 5f , the Apo-Si Conjugate manifestedefficient uptake through the cell membranes and accumulation, asdesired, within the cytoplasm.

Example 5b Murine B16 Melanoma Cells

The objective was to determine the capability of a Conjugate, comprisingtwo Apo-Si MNMs (each attached at a 5′-end of the strand), to performuptake into cultured B16 murine-skin melanoma cells. For this purpose,B16 cells were grown and maintained as described in Example 5a. Briefly,cells were grown in DMEM (Sigma Aldrich, USA), supplemented with 10%FBS, 2 mM L-glutamine and 1% Pen-Strep at 37° C., in a humidifiedincubator containing 5% CO₂. One day before transfection, 2×10⁴ B16cells were plated in standard 24-well plate chambers. 40 nM ofCy3-labeled 58-mer double-stranded DNA, conjugated to the Apo-si-11 MNMswere incubated with the cells for 24 hours in the presence of completegrowth medium. An identical Cy-3-labeled oligonucleotide, devoid of theApo-Si MNMs, was used as control, and was incubated with the cells forthe same time-period. Each well was washed twice with HBSS beforequantification of Fluorescence. Microscopy figures were taken with anOlympus BX51 microscope, as described above.

The B16 cells were also subjected to FACS analysis. For this purpose,one day before transfection, 16×10⁴ B16 cells were seeded in standard6-well plates. Ten and 40 nM of Cy3-labeled 58-mer dsDNA, conjugated totwo Apo-si-11 MNMs were incubated for 24 hours with complete growthmedium. A Cy3-labeled 58-mer DNA, devoid of the MNMs was used ascontrol. Cells were washed with HBSS, and analyzed for fluorescenceintensity with the BD FACSAria™ III as described above.

In addition, confocal microscopy was used, in order to further confirmuptake and cytoplasmic localization of the Apo-Si MNM conjugate,comprising the two MNMs. Cells were prepared as described above. Nuclearstaining with the Hoechst 33258 dye (Sigma Aldrich, USA, 1:1000 in HBSSfor about an hour) was also performed.

Marked uptake was detected in cells treated with the Apo-Si MNMConjugate comprising 58-mer double-stranded DNA, but not in the cellsexposed to an identical Cy3-labeled oligonucleotide but devoid of MNMs.This was evident in the fluorescent microcopy (FIG. 6a ), as well as inthe FACS analysis (FIG. 6b ). At 40 nM, the Apo-Si MNM Conjugatemanifested uptake by 98% percent of cells. A clear dose-response wasobserved, comparing signal intensities at 40 nM versus 10 nM. Confocalmicroscopy (FIG. 6c ) further showed efficient uptake of the Apo-SiConjugate through cell membranes, and accumulation in the cytoplasm.

Thus, Apo-Si MNM enables efficient delivery of a 58-mer ds-DNAoligonucleotide into B16 melanoma cells line, in adose-dependent-manner.

Example 5c C26 Murine Colon Adenocarcinoma Cells

In order to demonstrate the capability of Apo-Si MNMs, to enabledelivery of heavily-charged 58-mer dsDNA into C26 colon adeno-carcinomacells, cells were grown and maintained as described above. Briefly,cells were grown in DMEM, supplemented with 10% FBS 2 mM L-glutamine and1% Pen-Strep, at 37° C. in a humidified incubator, containing 5% CO2.

Cells were subjected to FACS analysis. For this propose, one day beforetransfection, 16×10⁴ C26 cells were seeded in a standard 6-well plates.40 nM of the 58-mer double-stranded DNA, conjugated to two Apo-Si MNMs,each at a 5′-end of the oligonucleotide, and linked to the Cy3fluorophore, were incubated for 24 hours in the presence of completegrowth medium. The same construct, devoid of the Apo-Si MNMs, served asControl. Cells were washed with HBSS and analyzed for fluorescenceintensity with the BD FACSAria™ III as mentioned above.

As shown in FIG. 7, marked Cy3 fluorescence was detected in 98% of cellstreated with the Apo-Si Conjugate. Such uptake was not detected in thecells exposed to the control oligonucleotide. Therefore, the Apo-Si MNMsenabled efficient trans-membrane delivery of the oligonucleotide.

Example 5d Human HeLa Cell Line

The objective was to demonstrate the capability of Apo-Si MNMs to enabledelivery of heavily-charged 58-mer dsDNA into HeLa human cervicalepithelial carcinoma cell line. For this purpose, cells were grown andmaintained as described above. Briefly, cells were grown in DMEMsupplemented with 10% FBS 2 mM L-glutamine and 1% Pen-Strep at 37° C.,in a humidified incubator, containing 5% CO₂.

For the FACS analysis, one day before transfection, 16×10⁴ HeLa cellswere seeded in standard 6-well plates. 40 nM of Cy3 labeled, 58-merdouble-stranded DNA, conjugated to two Apo-Si MNMs were incubated for 24hours in the presence of complete growth medium. Cy3-labeled 58-mer DNAwas used as control. Cells were washed with HBSS and analyzed forfluorescence intensity with the BD FACSAria™ III system, as mentionedabove. Cells, treated with 58-mer double stranded DNA, conjugated toApo-Si MNM manifested marked uptake into nearly all cells in the culture(FIG. 8). By contrast, such uptake was not observed in the cells treatedby the Control oligonucleotide. Therefore, in conclusion, Cy3 labeled,58-mer double-stranded DNA, thus carrying 58 negative charges, andconjugated to two Apo-Si MNMs manifests efficient delivery into culturedhuman HeLa cell line.

Taken together, these results presented in Example 5, and obtained fromfour distinct cell types: 3T3 murine fibroblast cells, murine melanomaB16 cells, murine C26 colon carcinoma cells, and human HeLa uterinecervical carcinoma cells, demonstrate an efficient trans-membranedelivery and uptake of highly-charged macromolecules when linked to oneor two Apo-Si MNMs. Such uptake was not observed in the controloligonucleotides, devoid of the MNMs. These data support the notion thatthe performance of the MNMs of the invention in enabling trans-membranedelivery of oligonucleotides is universal, and is not limited to aspecific cell type.

Example 6 A Mechanism for Intracellular Entrapment of siRNA, ComprisingAdministration of a Dicer Substrate

In an embodiment of the invention, it discloses a method for entrapmentof siRNA in the cytoplasm following its successful trans-membranedelivery by the Conjugates of the invention. The method is based on theactivity of the enzyme Dicer, an endocnulease which is capable ofprocessing double-stranded RNA, by cutting it at the size of 19-21 basepairs, suitable for interaction with RISC (RNA Inducible SilencingComplex) for gene silencing. Said method comprises: (i). Administrationof a Conjugate of the invention, wherein the oligonucleotide is a Dicersubstrate, consisting of a double-stranded RNA of 25-30-nucleotide long,being of the sequence required for silencing a specific target gene; andconjugated to MNMs of the invention, attached each at the 3′-end of thesense (passenger) strand, and/or at the 5′-end antisense (guide) strand;(ii). Trans-membrane delivery of the siRNA, enabled by the MNMs; (iii).Cleavage of the dsRNA by the Dicer enzyme, thus removing one MNM fromthe Duplex; (iv) physiological subsequent separation of the double-helix(e.g., by the Helicase enzyme) leading to release of the antisensestrand, to interact with RISC, in order to silence the specific targetgene (FIG. 3).

In order to examine cleavage by Dicer in vitro, siRNA duplexes (100pmol) were incubated in 20 ml of 20 mM Tris pH 8.0, 200 mM NaCl, 2.5 mMMgCl2, with 1 unit of recombinant human Dicer (Stratagene) for 24 h. A3-ml aliquot of each reaction (15 pmol RNA) was then separated in a 15%non-denaturing polyacrylamide gel, stained with GelStar (Ambrex) andvisualized using UV excitation. Electrospray-ionization liquidchromatography mass spectroscopy (ESILCMS) of the duplex RNAs before andafter treatment with Dicer was then performed, utilizing an Oligo HTCSsystem (Novatia), consisting of ThermoFinniganTSQ7000, Xcalibur datasystem, ProMass data processing software and Paradigm MS4 HPLC (MichromBioResources).

Example 7 Silencing of the EGFP Gene by a Conjugate of the Invention InVitro

The biological system used for this demonstration is human HeLA cells,stably expressing the enhanced green fluorescent protein (EGFP) gene(NIH-HeLa EGFP cells). The administered Conjugate of the Inventioncomprised siRNA, designed to silence the expression of the EGFP gene.Normally, unless utilizing a transfection reagent, such RNA constructcannot pass through the cell membrane into the cytoplasm, where it canexert its gene-silencing activity. Due to conjugation of this siRNA tothe MNMs of the invention [for example without limitation, E moietieshaving the structure as set forth in Formula (X)] gene silencingactivity is enabled and observed, without the need for a transfectionreagent.

For this purpose, cells were incubated with a Conjugate of theinvention, comprising siRNA designed for silencing of the EGFP protein(IDT, Iowa, USA), linked to two MNMs according to Formula (XVI). Thesequence of the double-stranded RNA was: Sense sequence 5′ to 3′:ACCCUGAAGUUCAUCUGCACCACCG (SEQ ID NO: 3); Antisense sequence 5′ to 3′:CGGUGGUGCAGAUGAACUUCAGGGUCA (SEQ ID NO: 4). A respective double-strandedDNA sequence, linked to the MNM moiety served as Control, since such DNAconstruct cannot exert gene-silencing activity.

Specifically, one day before the experiment, NIH-HeLa EGFP cells at theexponential growth phase were plated in 24-well plates, at a density of4.5×10⁴ cells/well, with DMEM and supplements growth medium (500μl/well), without antibiotics. The siRNA-Apo-Si-MNM Conjugate wasdiluted in 100 μl/well of Opti-Mem (Life technologies), and added to thecells, at a final concentration of 40 nM.

Gene silencing was assessed at 96 hours of incubation. At thattime-point, cells were washed with Hank's Buffered Salt Solution (HBSSbuffer; Biological Industries, Israel) and subjected to analysis.Detection and quantification of the EGFP-related fluorescent signal wasperformed by ELISA reader, utilizing Tecan Infinite® 200 PRO multimodereader (excitation wave length 488±4.5 nm and emission 535±10 nm). Asshown in FIG. 9, while the Conjugate comprising DNA did not show anysignificant silencing of the EGFP gene; gene silencing was exerted bythe respective Conjugate of siRNA linked to the MNMs.

Example 8 Delivery Across Cell Membranes of a Conjugate of the Inventionwhere E has the Structure According to Formula (VIIa)

3T3 cells and C26 cells were grown and prepared as described in Example5 above. Cells were incubated for 1, 2, and 24 hours with a Conjugatecomprising a 58-mer double-stranded (ds)DNA, linked to Cy3 fluorophore,and to two E moieties according to Formula (VIIa) wherein g=4. Twoconcentrations of the Conjugate were tested: 40 nM and 100 nM. Analysiscomprised fluorescent microscopy, and signal quantification by ELISAreader, as described in Example 5 above. An identical 58-mer dsDNA, notlinked to E moieties, served as Control.

Fluorescent detection of the Conjugate within the cells was possiblealready after one hour. Signal was obtained, as desired, in thecytoplasm. Signal intensity marked increased by 2 hours, with additionalaugmentation by 24 hours of incubation. Uptake was very clearly measuredby the ELISA reader. The ratios of signal intensity of the Conjugateversus the respective control dsDNA, devoid of the MNMs were, for theC26 cells: 352- and 320-fold; while for the 3T3, ratios were 104-, and101-fold, for concentrations of 40 nM and 100 nM, respectively.Therefore, for both cell types, the Conjugate of the invention enabledhighly efficient delivery of a highly-charged 58-mer ds-DNA, incomparison to the controls, devoid of the MNM moieties. Notable are alsothe observed dose-response, and the observation that the uptake was notsaturable, at least not at the examined dose-range.

Example 9 Mechanism of Redox-Sensitive Cleavage of the Conjugate of theInvention According to Formula (XI), and its Utilization for Targetingthe Cargo Drug (D) to the Cytoplasm

The mechanism is presented in a non-limiting manner. The Conjugate has adisulfide moiety within a six-member ring. Due to the oxidativeconditions that prevail in the extracellular space, this ring willmanifest stability in the plasma and extracellular space. By contrast,within the cells, the Conjugate will be subjected to reductive ambientconditions, provided by the high glutathione levels in the cytoplasm.Consequentially, there is cleavage of the disulfide bond, resulting infree thiol groups. Based on analogy to other disulfide molecules the pKavalues of the free thiol groups are anticipated to be about 8 and 9.Considering the physiological intracellular pH, being about 7, the vastmajority of the thiol groups generated upon cleavage of the disulfidebond, at any time, about 10-20% of the molecules will be as free thiolgroups (—SH), and not as the respective nucleophilic thiolate (—S⁻). TheConjugate according to Formula (XI) comprises an aromatic amide group.Importantly, this group is linked to the phenolic ring of estradiol(Ring A), which is therefore considered to be a good leaving group.Strategically, the amide carbonyl group is located five and four atomsaway from the thiol groups. Similar to its action in catalysis ofproteolysis in cysteine proteases, a nucleophilic action in attackingthe carbonyl carbon atom of the amide group, leading to cleavage of theestradiol moiety. This action therefore selectively liberates the cargodrug (D) in the cytoplasm. In the case that D is, for example, an siRNAor a Dicer Substrate, this will lead to entrapment of the highlynegatively-charged oligonucleotide in the cytoplasm, ready to interactin situ with the RNA-inducible silencing complex (RISC), in order toexert its gene silencing activity.

This mechanism is described in FIG. 10, where I. represents the intactConjugate n the extracellular space; II. represents the cleavage of thedisulfide bonfd in the reductive cytoplasmatic millieu; III. representsactivation of the thiol into the thiolate, in a pka-dependent process;IV. represents nucleophilic attack of the thiolate on the carbonylmoiety of the amide group; and V. represents the consequent cleavage ofthe Conjugate.

The invention claimed is:
 1. A method for delivery of a drug acrossbiological membranes, the method comprising utilization of a Conjugate,having the structure as set forth in Formula (I):

wherein: D is a drug to be delivered across biological membranes,selected from a group consisting of a protein, and a native or modified,single-stranded or double-stranded DNA or RNA, siRNA or ASO; y, z and ware each an integer, independently selected from 0, 1, 2, 3, 4, 5, 6;wherein at least one of y, z or w is different from 0; E, E′, or E″ canbe the same or different, each having the structure as set forth ingeneral Formula (II):(A)_(a)-B-L₁-Q₁-L₂-Q₂-L₃   Formula (II) wherein B is a steroid moietyselected from the group consisting of cholesterol, bile acid, estradiol,estriol; and any combination thereof; wherein B is optionally furthersubstituted by one or more groups selected from the group consisting of:linear C₁, C₂, C₃, C₄, C₅, C₆,C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄alkyl,cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄,alkyl or hetero-alkyl; linear C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃, C₁₄, alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄ alkylene or heteroalkylene; C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄ aryl or heteroaryl; wherein B is optionallysubstituted by a hydroxyl, amine, or thiol; Q₁ and Q₂ are eachindependently an optionally cleavable group, selected from null, ester,thio-ester, amide [—C(═O)—NH— or —NH—C(═O)—], carbamate [—O—C(═O)—NH— or—NH—C(═O)—O—], urea [—NH—C(═O)—NH—], disulfide [—(S—S)—], ether [—O—],imidazole, triazole, analogs of BAPTA and EGTA; and any combinationsthereof; L₁and L₂ are each independently selected from null or L₁, L₂and L₃ are each independently selected from the group consisting of:linear C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃ or C₁₄,alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂,C₁₃, C₁₄, alkyl or hetero-alkyl; linear, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃ or C₁₄ alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇,C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄alkylene or heteroalkylene; C₅, C₆, C₇,C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃ or C₁₄ aryl or heteroaryl; —(O—CH₂—CH₂)_(u)—,wherein u is an integer of 1, 2, 3, 4 or 5; nucleoside, nucleotide;imidazole, azide, acetylene; and any combinations thereof; wherein eachgroup is optionally substituted by one or more hydroxyl, amine, orthiol; wherein each of Q₁, Q₂, L₁, L₂ and L₃ is optionally an initiatorgroup, selected from C₄,C₅,C₆-1,2-dithiocycloalkyl(1,2-dithiocyclo-butane;1,2-dithiocyclo-pentane; 1,2-dithiocyclohexane; 1,2-dithiocycloheptane);γ-Lactam (5 atoms amide ring), δ-Lactam (6 atoms amide ring) or ε-Lactam(7 atoms amide ring); γ-butyrolactone (5 atoms ester ring),δ-valerolactone (6 atoms ester ring) or ε-caprolactone (7 atoms esterring); an amine group or both an initiator group and an amine group; Ais selected from the group consisting of Formulae (III), Formula (IV),Formula (V) and Formula (VI):

M is selected from —O— or —CH₂—; and g, h and k are each individually aninteger selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 and 16; * is —H, or a point of linkage to B; ais an integer, selected from 1, 2, 3 or
 4. 2. A method according toclaim 1, wherein y=1, z=o and w=0; or y=1, z=1 and w=0.
 3. A method fordelivery of a drug across biological membranes, the method comprisingutilization of a Conjugate, having the structure as set forth in Formula(I):

wherein: D is a drug to be delivered across biological membranes,selected from a group consisting of a protein, and a native or modified,single-stranded or double-stranded DNA or RNA, siRNA or ASO; y, z and ware each an integer, independently selected from 0, 1, 2, 3, 4, 5, 6;wherein at least one of y, z or w is different from 0; E, E′ or E″ hasthe structure as set forth in Formula (VII):

wherein k is an integer, selected from 0, 1, 2, 3, and 4; U is selectedfrom null, O, N and NH; and wherein B is a steroid moiety selected fromthe group consisting of cholesterol, bile acid, estradiol, estriol; andany combination thereof; wherein B is optionally further substituted byone or more groups selected from the group consisting of: linear C₁, C₂,C₃, C₄, C₅, C₆,C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄alkyl, cyclic orbranched C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, alkyl orhetero-alkyl; linear, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄ alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃, C₁₄alkylene or heteroalkylene; C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃ , C₁₄ aryl or heteroaryl; wherein B is optionally substitutedby a hydroxyl, amine, or thiol; Q₁ and Q₂ are each independently anoptionally cleavable group, selected from null, ester, thio-ester, amide[—C(═O)—NH— or —NH—C(═O)—], carbamate [—O—C(═O)—NH— or —NH—C(═O)—O—],urea [—NH—C(═O)—NH—], disulfide [—(S—S)—], ether [—O—], imidazole,triazole, analogs of BAPTA and EGTA; and any combinations thereof; L₁andL₂ are each independently selected from null or L₁, L₂ and L₃ are eachindependently selected from the group consisting of: linear, C₁, C₂, C₃,C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃ , C₁₄, alkyl, cyclic orbranched C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃ or C₁₄, alkyl orhetero-alkyl; linear, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄ alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃or C₁₄alkylene or heteroalkylene; C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃ or C₁₄ aryl or heteroaryl; —(O—CH₂—CH₂)_(u)—, wherein u is aninteger of 1, 2, 3, 4 or 5; nucleoside, nucleotide; imidazole, azide,acetylene; and any combinations thereof; wherein each group isoptionally substituted by one or more hydroxyl, amine, or thiol; whereineach of Q₁, Q₂, L₁, L₂ and L₃ is optionally an initiator group, selectedfrom C₄, C₅,C₆-1,2-dithiocycloalkyl(1,2-dithiocyclo-butane;1,2-dithiocyclo-pentane; 1,2-dithiocyclohexane; 1,2-dithiocycloheptane);γ-Lactam (5 atoms amide ring), δ-Lactam (6 atoms amide ring) or ε-Lactam(7 atoms amide ring); γ-butyrolactone (5 atoms ester ring),δ-valerolactone (6 atoms ester ring) or ε-caprolactone (7 atoms esterring); an amine group; or both an initiator group and an amine group. 4.A method according to claim 3, wherein k=1.
 5. A method according toclaim 3, wherein L₁and L₂ are each individually selected form null orL₁, L₂ and L₃ are each individually selected from null and a linear C₁,C₂, C₃, C₄, C₅, C₆, C₇, C₈ cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈hydrocarbon chain; L₁, L₂ and L₃ can be the same or different.
 6. Amethod according to claim 3, wherein Q₁ or Q₂ is selected from the groupconsisting of an amide group, an ester, a carbamate, a disulfide,imidazole; and any combination thereof.
 7. A method according to claim3, wherein L₁, L₂ or L₃ is 1,2-dithiocyclo-butane, optionally linked toan amide or amine group.
 8. A method according to claim 1, wherein E, E′or E″ have the structure as set forth in Formulae (VIIa):

wherein g stands for an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13,
 14. 9. A method according to claim 8, wherein g=2.
 10. A methodaccording to claim 1, wherein E, E′ or E″ have the structure as setforth in Formula (VIII):

where k stands for an integer, selected from the group consisting of 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16; h stands foran integer, selected from the group consisting of 0, 1, 2, 3 and 4; U isselected from null, O, N or NH; Z is selected from hydrogen, hydroxyland amine groups; Y is selected from —CH—and a nitrogen atom.
 11. Amethod according to claim 10, wherein k=1, and h=1.
 12. A methodaccording to claim 1, wherein E, E′ or E″ have the structure as setforth in Formula (IX):

or has the conjugate structure as set forth in Formula (X):

wherein w stands for an integer of 0, 1, 2 or 3; t stands for an integerof 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14; p stands for aninteger of 0, 1, 2 or 3; R₁ stands for hydrogen, or an alkyl group of 1,2 or 3 carbon atoms; or has the conjugate structure as set forth inFormula (XI):

or has the conjugate structure as set forth in Formula (XII):

wherein b stands for an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; J isselected from null, nitrogen or an oxygen atom; or has the conjugatestructure as set forth in Formula (XIII):

or has the conjugate structure as set forth in-Formula (XIV):

or has the conjugate structure as set forth in Formula (XV):

where y stands for an integer, selected from the group consisting of 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12; or has the conjugate structureas set forth in Formula (XVI):

or has the conjugate structure as set forth in Formula (XVII):

where y stands for an integer, selected from the group consisting of 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and
 12. In a preferred embodiment,y=1.
 13. A conjugate according to Formula (I):

wherein: D is a drug to be delivered across biological membranes,selected from a group consisting of a protein, and a native or modified,single-stranded or double-stranded DNA or RNA, siRNA or ASO; y, z and ware each an integer, independently selected from 0, 1, 2, 3, 4, 5, 6;wherein at least one of y, z or w is different from 0; E, E′, or E″ canbe the same or different, each having the structure as set forth ingeneral Formula (II):(A)_(a)-B-L₁-Q₁-L₂-Q₂-L₃   Formula (II) wherein B is a steroid moietyselected from the group consisting of cholesterol, bile acid, estradiol,estriol; and any combination thereof; wherein B is optionally furthersubstituted by one or more groups selected from the group consisting of:linear C₁, C₂, C₃, C₄, C₅, C₆,C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄alkyl,cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄,alkyl or hetero-alkyl; linear, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃ , C₁₄ alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄alkylene or heteroalkylene; C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃ , C₁₄ aryl or heteroaryl; wherein B is optionallysubstituted by a hydroxyl, amine, or thiol; Q₁ and Q₂ are eachindependently an optionally cleavable group, selected from null, ester,thio-ester, amide [—C(═O)—NH— or —NH—C(═O)—], carbamate [—O—C(═O)—NH— or—NH—C(═O)—O—], urea [—NH—C(═O)—NH—], disulfide [—(S—S)—], ether [—O—],imidazole, triazole, analogs of BAPTA and EGTA; and any combinationsthereof; L₁and L₂ are each independently selected from null or L₁, L₂and L₃ are each independently selected from the group consisting of:linear, C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃ , C₁₄,alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂,C₁₃or C₁₄, alkyl or hetero-alkyl; linear C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇,C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃or C₁₄ alkylene or heteroalkylene; C₅, C₆, C₇,C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃or C₁₄ aryl or heteroaryl; —(O—CH₂—CH₂)_(u)—,wherein u is an integer of 1, 2, 3, 4 or 5; nucleoside, nucleotide;imidazole, azide, acetylene; and any combinations thereof; wherein eachgroup is optionally substituted by one or more hydroxyl, amine, orthiol; wherein each of Q₁, Q₂, L₁, L₂ and L₃ is optionally an initiatorgroup, selected from C₄,C₅,C₆-1,2-dithiocycloalkyl(1,2-dithiocyclo-butane;1,2-dithiocyclo-pentane; 1,2-dithiocyclohexane; 1,2-dithiocycloheptane);γ-Lactam (5 atoms amide ring), δ-Lactam (6 atoms amide ring) or ε-Lactam(7 atoms amide ring); γ-butyrolactone (5 atoms ester ring),δ-valerolactone (6 atoms ester ring) or ε-caprolactone (7 atoms esterring); an amine group; or both an initiator group and an amine group; Ais selected from the group consisting of Formulae (III), Formula (IV),Formula (V) and Formula (VI):

M is selected from —O—or -CH₂-; and g, h and k are each individually aninteger selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 and 16; * is —H, or a point of linkage to B; ais an integer, selected from 1, 2, 3 or
 4. 14. A conjugate according toclaim 13, wherein the drug is a macromolecule, selected from the groupconsisting of siRNA, ASO and a therapeutic protein.
 15. A conjugateaccording to claim 14, wherein the therapeutic protein comprises aCRISPR protein.
 16. A conjugate according to claim 15, wherein theCRISPR protein is Cas9 protein.
 17. A pharmaceutical composition,comprising a Conjugate according to claim 13 and apharmaceutically-acceptable salt or carrier.
 18. A method for deliveryof a drug into biological cells, wherein said cells are in culture, orin a living animal or a human subject; the method comprising contactingthe cells with a Conjugate according to claim
 13. 19. The methodaccording to claim 1, where the biological membrane is selected from agroup consisting of cell membranes and biological barriers, selectedfrom the blood-brain-barrier, blood-ocular-barrier or theblood-fetal-barrier.
 20. A precursor, having the structure as set forthin Formula (XVII):

wherein W is a moiety, selected from E, E′ or E″as described in any ofFormula II, VII, VIIa, VIII, IX, X, XI, XII, XIII, XIV, XV or XVI;wherein Formula (II) is defined as:(A)_(a)-B-L₁-Q₁-L₂-Q₂-L₃   Formula (II) wherein B is a steroid moietyselected from the group consisting of cholesterol, bile acid, estradiol,estriol; and any combination thereof; wherein B is optionally furthersubstituted by one or more groups selected from the group consisting of:linear C₁, C₂, C₃, C₄, C₅, C₆,C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄alkyl,cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄,alkyl or hetero-alkyl; linear, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃ , C₁₄ alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄alkylene or heteroalkylene; C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃ , C₁₄ aryl or heteroaryl; wherein B is optionallysubstituted by a hydroxyl, amine, or thiol; Q₁ and Q₂ are eachindependently an optionally cleavable group, selected from null, ester,thio-ester, amide [—C(═O)—NH— or —NH—C(═O)—], carbamate [—O—C(═O)—NH— or—NH—C(═O)—O—], urea [—NH—C(═O)—NH—], disulfide [—(S—S)—], ether [—O—],imidazole, triazole, analogs of BAPTA and EGTA; and any combinationsthereof; L₁and L₂ are each independently selected from null or L₁, L₂and L₃ are each independently selected from the group consisting of:linear, C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃ , C₁₄,alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂,C₁₃, C₁₄, alkyl or hetero-alkyl; linear C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇,C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃or C₁₄ alkylene or heteroalkylene; C₅, C₆, C₇,C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃or C₁₄ aryl or heteroaryl; —(O—CH₂—CH₂)_(u)—,wherein u is an integer of 1, 2, 3, 4 or 5; nucleoside, nucleotide;imidazole, azide, acetylene; and any combinations thereof; wherein eachgroup is optionally substituted by one or more hydroxyl, amine, orthiol; wherein each of Q₁, Q₂, L₁, L₂ and L₃ is optionally an initiatorgroup, selected from C₄,C₅,C₆-1,2-dithiocycloalkyl(1,2-dithiocyclo-butane;1,2-dithiocyclo-pentane; 1,2-dithiocyclohexane; 1,2-dithiocycloheptane);γ-Lactam (5 atoms amide ring), δ-Lactam (6 atoms amide ring) or ε-Lactam(7 atoms amide ring); γ-butyrolactone (5 atoms ester ring),δ-valerolactone (6 atoms ester ring) or ε-caprolactone (7 atoms esterring); an amine group; or both an initiator group and an amine group; Ais selected from the group consisting of Formulae (III), Formula (IV),Formula (V) and Formula (VI):

M is selected from —O—or —CH₂-; and g, h and k are each individually aninteger selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 and 16; * is —H, or a point of linkage to B; ais an integer, selected from 1, 2, 3 or 4 and wherein Formula VII isdefined as:

wherein k is an integer, selected from 0, 1, 2, 3, and 4; U is selectedfrom null, O, N and NH; D is a drug to be delivered across biologicalmembranes, selected from a group consisting of a protein, and a nativeor modified, single-stranded or double-stranded DNA or RNA, siRNA orASO; Formulae (VIIa) is defined as:

wherein g stands for an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 Formula (VIII) is defined as:

where k stands for an integer, selected from the group consisting of 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16; h stands foran integer, selected from the group consisting of 0, 1, 2, 3 and 4; U isselected from null, O, N or NH; Z is selected from hydrogen, hydroxyland amine groups; Y is selected from —CH— and a nitrogen atom Formula(IX) is defined as:

Formula (X) is defined as:

wherein w stands for an integer of 0, 1, 2 or 3; t stands for an integerof 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14; p stands for aninteger of 0, 1, 2 or 3; R₁ stands for hydrogen, or an alkyl group of 1,2 or 3 carbon atoms; Formula (XI) is defined as:

Formula (XII) is defined as:

wherein b stands for an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; J isselected from null, nitrogen or an oxygen atom; Formula (XIII) isdefined as:

Formula (XIV) is defined as:

Formula (XV) is defined as:

where y stands for an integer, selected from the group consisting of 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12; Formula (XVI) is defined as:

or has a structure as set forth in Formula (XVIII):

wherein G is a moiety, selected from E, E′ or E″ as described in any ofFormula II, VII, VIIa, VIII, IX, X, XI, XII, XIII, XIV, XV or XVI andwhere DMT (dimethoxytrityl) is a protecting group for a hydroxyl; andCPG is Controlled Pore Glass.
 21. A precursor, having the structure asset forth in any of Formula II, VII, VIIa, VIII, IX, X, XI, XII, XIII,XIV, XV or XVI, wherein D is selected from phosphoroamidate, anactivated ester, azide or acetylene and wherein Formula (II) is definedas:(A)_(a)-B-L₁-Q₁-L₂-Q₂-L₃   Formula (II) wherein B is a steroid moietyselected from the group consisting of cholesterol, bile acid, estradiol,estriol; and any combination thereof; wherein B is optionally furthersubstituted by one or more groups selected from the group consisting of:linear C₁, C₂, C₃, C₄, C₅, C₆,C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄alkyl,cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄,alkyl or hetero-alkyl; linear, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃ , C₁₄ alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄alkylene or heteroalkylene; C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃ , C₁₄ aryl or heteroaryl; wherein B is optionallysubstituted by a hydroxyl, amine, or thiol; Q₁ and Q₂ are eachindependently an optionally cleavable group, selected from null, ester,thio-ester, amide [—C(═O)—NH— or —NH—C(═O)—], carbamate [—O—C(═O)—NH— or—NH—C(═O)—O—], urea [—NH—C(═O)—NH—], disulfide [—(S—S)—], ether [—O—],imidazole, triazole, analogs of BAPTA and EGTA; and any combinationsthereof; L₁and L₂ are each independently selected from null or L₁, L₂and L₃ are each independently selected from the group consisting of:linear C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃ , C₁₄,alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂,C₁₃or C₁₄, alkyl or hetero-alkyl; linear C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, alkyl, cyclic or branched C₃, C₄, C₅, C₆, C₇,C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃or C₁₄ alkylene or heteroalkylene; C₅, C₆, C₇,C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃or C₁₄ aryl or heteroaryl; —(O—CH₂—CH₂)_(u)—,wherein u is an integer of 1, 2, 3, 4 or 5; nucleoside, nucleotide;imidazole, azide, acetylene; and any combinations thereof; wherein eachgroup is optionally substituted by one or more hydroxyl, amine, orthiol; wherein each of Q₁, Q₂, L₁, L₂ and L₃ is optionally an initiatorgroup, selected from C₄,C₅,C₆-1,2-dithiocycloalkyl(1,2-dithiocyclo-butane;1,2-dithiocyclo-pentane; 1,2-dithiocyclohexane; 1,2-dithiocycloheptane);γ-Lactam (5 atoms amide ring), δ-Lactam (6 atoms amide ring) or ε-Lactam(7 atoms amide ring); γ-butyrolactone (5 atoms ester ring),δ-valerolactone (6 atoms ester ring) or ε-caprolactone (7 atoms esterring); an amine group; or both an initiator group and an amine group; Ais selected from the group consisting of Formulae (III), Formula (IV),Formula (V) and Formula (VI):

M is selected from —O—or —CH₂-; and g, h and k are each individually aninteger selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 and 16; * is —H, or a point of linkage to B; ais an integer, selected from 1, 2, 3 or 4 and wherein Formula VII isdefined as:

wherein k is an integer, selected from 0, 1, 2, 3, and 4; U is selectedfrom null, O, N and NH Formulae (VIIa) is defined as:

wherein g stands for an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 Formula (VIII) is defined as:

where k stands for an integer, selected from the group consisting of 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16; h stands foran integer, selected from the group consisting of 0, 1, 2, 3 and 4; U isselected from null, O, N or NH; Z is selected from hydrogen, hydroxyland amine groups; Y is selected from —CH—and a nitrogen atom Formula(IX) is defined as:

Formula (X) is defined as:

wherein w stands for an integer of 0, 1, 2 or 3; t stands for an integerof 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14; p stands for aninteger of 0, 1, 2 or 3; R₁ stands for hydrogen, or an alkyl group of 1,2 or 3 carbon atoms; Formula (XI) is defined as:

Formula (XII) is defined as:

wherein b stands for an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; J isselected from null, nitrogen or an oxygen atom; Formula (XIII) isdefined as:

Formula (XIV) is defined as:

Formula (XV) is defined as:

where y stands for an integer, selected from the group consisting of 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12; Formula (XVI) is defined as:

and wherein Formula (XVIII):

wherein G is a moiety, selected from E, E′ or E″ as described in any ofFormula II, VII, VIIa, VIII, IX, X, XI, XII, XIII, XIV, XV or XVI andwhere DMT (dimethoxytrityl) is a protecting group for a hydroxyl; andCPG is Controlled Pore Glass.
 22. The method according to claim 1,wherein the steroid moiety is selected from cholesterol, bile acid,estrogen, estradiol, and estriol.
 23. A method of delivering a drugacross a biological membrane, the method comprising: conjugating atleast one asymmetrically polar chemical moiety to a drug, wherein themoiety is capable of moving within the hydrophobic membrane milieutowards the membrane core, directed by the internal membrane electricalfield; and contacting the biological membrane with the conjugated-drug.24. A method of delivering a drug across a biological membrane, themethod comprising: providing a conjugate of a drug and at least oneasymmetrically polar chemical moiety, wherein the moiety is capable ofmoving within the hydrophobic membrane milieu towards the membrane core,directed by the internal membrane electrical field; and contacting thebiological membrane with the conjugated-drug.
 25. A conjugate of a drugand at least one asymmetrically polar chemical moiety, wherein themoiety is capable of moving within the hydrophobic membrane milieutowards the membrane core, directed by the internal membrane electricalfield, for use in delivering a drug across a biological membrane.
 26. Amethod according to claim 24, wherein the asymmetrically polar chemicalmoiety has an octanol/water partition coefficient >1.
 27. A methodaccording to claim 24, wherein the asymmetrically polar chemical moietycomprises a focused negative pole comprising at least oneelectronegative atom, and a dispersed positive pole.
 28. A methodaccording to claim 27, wherein the negative pole comprises at least oneelectronegative atom selected from a halogen such as fluorine, oroxygen, and/or the positive pole comprises a hydrocarbon chain(s), asteroid moiety or a combinations thereof, optionally substituted byelectropositive atoms selected from silicon, boron, phosphorus andsulfur.
 29. A conjugate according to claim 13, wherein E, E′ or E″ hasthe structure as set forth in Formula (VII):


30. A pharmaceutical composition, comprising a Conjugate according toclaim 29 and a pharmaceutically-acceptable salt or carrier.